Pulsed Electron Paramagnetic Resonance Techniques Research Articles

A Multi-Frequency Pulse EPR and ENDOR Approach to Study Strongly Coupled Nuclei in Frozen Solutions of High-Spin Ferric Heme Proteins

In spite of the tremendous progress in the field of pulse electron paramagnetic resonance (EPR) in recent years, these techniques have been scarcely used to investigate high-spin (HS) ferric heme proteins. Several technical and spin-system-specific reasons can be identified for this. Additional problems arise when no single crystals of the heme protein are available. In this work, we use the example of a frozen solution of aquometmyoglobin (metMb) to show how a multi-frequency pulse EPR approach can overcome these problems. In particular, the performance of the following pulse EPR techniques are tested: Davies electron nuclear double resonance (ENDOR), hyperfine correlated ENDOR (HYEND), electron-electron double resonance (ELDOR)-detected NMR, and several variants of hyperfine sublevel correlation (HYSCORE) spectroscopy including matched and SMART HYSCORE. The pulse EPR experiments are performed at X-, Q- and W-band microwave frequencies. The advantages and drawbacks of the different methods are discussed in relation to the nuclear interaction that they intend to reveal. The analysis of the spectra is supported by several simulation procedures, which are discussed. This work focuses on the analysis of the hyperfine and nuclear-quadrupole tensors of the strongly coupled nuclei of the first coordination sphere, namely, the directly coordinating heme and histidine nitrogens and the 17O nucleus of the distal water ligand. For the latter, 17O-isotope labeling was used. The accuracy of our results and the spectral resolution are compared in detail to an earlier single-crystal continuous-wave ENDOR study on metMb, and it will be shown how additional information can be obtained from the multi-frequency approach. The current work is therefore prone to become a template for future EPR/ENDOR investigations of HS ferric heme proteins for which no single crystals are available.

Electron paramagnetic resonance investigation of metalloendofullerene derived carbon nanotube peapods

Single Wall Carbon Nanotubes (SWCNT) prepared by the "super growth" method and arc-grown material were used as templates for peapod preparation with La@C(82). A qualitative change of the electron paramagnetic resonance (EPR) properties of La@C(82) is observed after incorporation into SWNT. The loss of lanthanum hyperfine interaction in combination with the observed increase of EPR susceptibility by two orders of magnitude after peapod preparation when comparing with signals from "empty" tubes is indicative for the generation of itinerant spins by charge and spin transfer from La@C(82) to the tubes. This interpretation is supported by the observation of fast spin dephasing, detected with pulsed EPR techniques.

High-Resolution Structure of a Na +/H + Antiporter Dimer Obtained by Pulsed Electron Paramagnetic Resonance Distance Measurements

Transient or partial formation of complexes between biomacromolecules is a general mechanism used to control cellular functions. Several of these complexes escape structure determination by crystallographic means. We developed a new approach for determining the structure of protein dimers in the native environment (e.g., in the membrane) with high resolution in cases where the structure of the two monomers is known. The approach is based on measurements of distance distributions between spin labels in the range between 2 and 6 nanometers by a pulsed electron paramagnetic resonance technique and explicit modeling of spin label conformations. By applying this method to the membrane protein homodimer of the Na +/H + antiporter NhaA of Escherichia coli, the structure of the presumably physiological dimer was determined. It reveals two points of contact between the two monomers, with one of them confirming results of earlier cross-linking experiments.

Mapping the molecular axes of pentacene-d 14 doped in p-terphenyl single crystal using pulsed EPR technique in near zero magnetic field

The pulsed electron paramagnetic resonance (EPR) technique using computer controlled three-dimensional (3D) near-zero fields (NZF, 0–1.5 mT) have previously been reported. This paper reports on a comprehensive NZF pulsed EPR study of pentacene-d 14 doped in p-terphenyl (PDPT) single crystals. Molecular symmetry axes are determined from the analyses of these NZF EPR spectra.

Spin multiplicity and charge state of a silicon vacancy(TV2a)in4H-SiC determined by pulsed ENDOR

In this paper, we unambiguously re-determine the spin multiplicity of ${T}_{V2a}$ by pulsed electron nucleus double resonance technique. The ${T}_{V2a}$ center is one of the most commonly observed defects in $4H$-SiC, and its origin was identified as one belonging to a class of negatively charged silicon vacancy by means of continuous-wave electron paramagnetic resonance (EPR) and the two-dimensional nutation method of pulsed EPR technique. However, a model with the spin multiplicity of triplet $(S=1)$ and the neutral charge state has recently been suggested. Our result clearly shows that ${T}_{V2a}$ is a quartet spin $(S=3∕2)$ state and thus should be single-negatively charged $(\ensuremath{-}1)$.

UTILIZATION OF POLARIZED ELECTRON SPIN OF ORGANIC MOLECULES IN QUANTUM COMPUTING

The possibility of utilizing highly polarized electron spin of the photo-excited triplet state of organic semiconductors (pentacene molecules) embedded in organic crystals and mesoporous materials by zero-field (ZF) and near zero-field (NZF) pulsed electron paramagnetic resonance (EPR) techniques in a quantum computer will be explored. A simple logic gate, such as CNOT, utilizing such highly polarized electron spins communicating with the surrounding paramagnetic nuclei via hyperfine interaction will be discussed. Major advantages of these approaches are: (1) high electron spin polarization, (2) possible single-molecule detection, (3) orchestrated quantum perturbations can be imposed, and (4) pulsed ZF and NZF EPR techniques can be performed without high magnetic field.

Electron Spin Coherence of Phosphorus Donors in Isotopically Purified 29Si

We investigate spin coherence time of electrons bound to phosphorus donors in silicon single crystals, employing a pulsed electron paramagnetic resonance technique. The samples were isotopically controlled so that they may possess different concentrations (about 5% and 100%) of 29Si, which is the only non-zero-spin (spin-1/2) stable isotope of Si. Both 29Si-concentration dependence and orientation dependence of the electron spin coherence time demonstrate that the decoherence is caused by spectral diffusion due to mutual flip-flops of the environmental nuclear spins. The detail analysis of spin echo decay curves enables the unique assignment of the host sites responsible for electron spin echo envelope modulation.

Relaxation filtered hyperfine (REFINE) spectroscopy: a novel tool for studying overlapping biological electron paramagnetic resonance signals applied to mitochondrial complex I.

A simple strategy to separate overlapping electron paramagnetic resonance (EPR) signals in biological systems is presented. Pulsed EPR methods (inversion- and saturation-recovery) allow the determination of the T(1) spin-lattice relaxation times of paramagnetic centers. T(1) may vary by several orders of magnitude depending on the species under investigation. These variations can be employed to study selectively individual species from a spectrum that results from an overlap of two species using an inversion-recovery filtered (IRf) pulsed EPR technique. The feasibility of such an IRf field-swept technique is demonstrated on model compounds (alpha,gamma-bisphenylene-beta-phenylallyl-benzolate, BDPA, and 2,2,6,6-tetramethyl-piperidine-1-oxyl, TEMPO) and a simple strategy for the successful analysis of such mixtures is presented. Complex I is a multisubunit membrane protein of the respiratory chain containing several iron-sulfur (FeS) centers, which are observable with EPR spectroscopy. It is not possible to investigate the functionally important FeS cluster N2 separately because this EPR signal always overlaps with the other FeS signals. This cluster can be studied selectively using the IRf field-swept technique and its EPR spectrum is in excellent agreement with previous cw-EPR data from the literature. In addition, the possibility to separate the hyperfine spectra of two spectrally overlapping paramagnetic species is demonstrated by applying this relaxation filter together with hyperfine spectroscopy (REFINE). For the first time, the application of this filter to a three-pulse electron spin-echo envelope modulation (ESEEM) pulse sequence is demonstrated to selectively observe hyperfine spectra on a system containing two paramagnetic species. Finally, REFINE is used to assign the observed nitrogen modulation in complex I to an individual iron-sulfur cluster.

Dynamics, Site Binding, and Distribution of Counterions in Polyelectrolyte Solutions Studied by Electron Paramagnetic Resonance Spectroscopy

A microscopic picture of counterion condensation in liquid and glassy frozen solutions of the cationic polyelectrolyte poly(diallyldimethylammonium chloride) (PDADMAC) is derived from data obtained by a combination of continuous-wave and pulse electron paramagnetic resonance techniques. The condensation of such divalent anions to the polyelectrolyte chain in the presence of a large excess of polyelectrolyte and monovalent counterions can be described by a dynamic equilibrium between specifically site-bound and nonspecifically territorially bound counterions with exchange between the two states proceeding on time scales significantly shorter than 1 ns. No free divalent counterions are detected. The dynamic electrostatic attachment is manifest in an axially symmetric rotational diffusion tensor, with the unique axis of fast rotation corresponding to the electrostatic bond between one sulfonate group of Fremy's salt and the quaternary ammonium group of the PDADMAC repeat unit. A distance of 0.43 nm between the electron spin and the 14N nucleus of the ammonium group is found by electron spin−echo envelope modulation spectroscopy, suggesting that the site-bound state corresponds to contact ion pairs. The same experiment provides an estimate of 20% site-bound and 80% territorially bound divalent counterions in glycerol/water glassy frozen solution. Pulse electron−electron double resonance measurements show that on nanometer length scales the counterions are virtually homogeneously distributed in three dimensions for high polyelectrolyte concentration but linearly distributed along stretched chains at low polyelectrolyte concentrations where no overlap of chains is expected.

Applications of pi-photon-induced transparency in two-frequency pulse electron paramagnetic resonance experiments.

An approach to pulse electron paramagnetic resonance (EPR) experiments which are based on two different resonance fields is introduced. Instead of using two microwave (mw) sources or a magnetic field jump, bichromatic pulses consisting of a transverse microwave field with frequency omega(mw) and a longitudinal radio frequency field with frequency omega(rf) are employed. Such bichromatic pulses excite a number of multiple photon transitions at frequencies omega(mw)+komega(rf) (k in Z). The pi-photon-induced transparency phenomenon is used to select the required transitions. This approach is used in the stimulated soft electron spin echo envelope modulation and the four-pulse double electron-electron resonance experiments. The results obtained using the bichromatic pulse approach are in agreement with those obtained with the standard pulse EPR techniques. It is shown that applying bichromatic pulses is straightforward and advantageous in several respects.

Continuous-wave and pulsed EPR study of the negatively charged silicon vacancy withS=32andC3vsymmetry inn-type4H−SiC

The ${T}_{V2a}$ center, which was suggested to be the excited triplet state $(S=1)$ of the neutral silicon vacancy related defect [S\"orman et al., Phys. Rev. B $61,$ 2613 (2000)] in the electron-irradiated n-type $4H\ensuremath{-}\mathrm{SiC}$ has been studied by continuous wave and pulsed electron paramagnetic resonance (EPR). The spin multiplicity of ${T}_{V2a}$ has been determined to be quartet $(S=3/2)$ by the nutation method of pulsed EPR technique. From the temperature dependence of the signal intensity, it has been revealed that the ${T}_{V2a}$ spectrum is arising from an electronic ground state. From the measurement of the ${}^{13}\mathrm{C}$ hyperfine interactions of the nearest neighbors which has been enabled by the selective enhancement of the ${T}_{V2a}$ signals through the spin polarization by a laser light (808 nm) illumination, the center is unambiguously identified to be a single silicon vacancy. It is proposed that the center is a negatively charged silicon vacancy of ${C}_{3v}$ symmetry with the crystal field distorted slightly from regular tetrahedron. The triply degenerate ${t}_{2}$ state of an electronic configuration ${a}_{1}^{2}{t}_{2}^{3}$ under ${T}_{d}$ symmetry splits into ${a}_{1}$ and e by the distortion to ${C}_{3v}.$ The high spin configuration $[{a}_{1}{e}^{2}$ or ${e}^{2}{a}_{1}]$ which reduces the electron repulsion energy is preferred rather than the low spin configuration expected from the symmetry-lowering crystal field alone. The important role of the many-electron effect in determining the ground-state configuration is demonstrated clearly by ${T}_{V2a}$ in which the electron-electron interactions (the electronic repulsion and the electron exchange) compete against the crystal-field splitting.

Relaxation mechanism in γ-ray-irradiated L-alanine studied by transfer saturation EPR and pulse EPR

Stable paramagnetic centers in γ-ray-irradiated L-alanine dosimeters exhibit a maximum in relaxation rate in the vicinity of 190 K. The mechanism of this relaxation rate has been investigated on the first stable alanine radical center, SARI, by employing continuous-wave transfer saturation electron paramagnetic resonance and pulse electron paramagnetic resonance techniques. The detected in-phase and out-of-phase spectra as well as phase memory times,T M, indicate that besides the well-known τp of the CH3 group of SAR1 an additional correlation time, τl (ΔElk=2689±50 K and 0 τ10 = 0.15 ± 0.03 ps), is involved in the transverse relaxation process and effects the SAR1 center. For the SAR1 center this mechanism originates from the hindered motion of undamaged CH3 and NH 3 + groups in the lattice. The motion of these groups additionally effects the spectrum of the SAR1 center through averaging out of the anisotropic splitting.

Simulations of the 1 H electron spin echo–electron nuclear double resonance and 2 H electron spin echo envelope modulation spectra of exchangeable hydrogen nuclei coupled to the S 2 –state photosystem II manganese cluster

The pulsed EPR methods of electron spin echo envelope modulation (ESEEM) and electron spin echo-electron nuclear double resonance (ESE-ENDOR) are used to investigate the proximity of exchangeable hydrogens around the paramagnetic S(2)-state Mn cluster of the photosystem II oxygen-evolving complex. Although ESEEM and ESE-ENDOR are both pulsed electron paramagnetic resonance techniques, the specific mechanisms by which nuclear spin transitions are observed are quite different. We are able to generate good simulations of both (1)H ESE-ENDOR and (2)H ESEEM signatures of exchangeable hydrogens at the S(2)-state cluster. The convergence of simulation parameters for both methods provides a high degree of confidence in the simulations. Several exchangeable protons-deuterons with strong dipolar couplings are observed. In the simulations, two of the close ( approximately 2.5 A) hydrogen nuclei exhibit strong isotropic couplings and are therefore most probably associated with direct substrate ligation to paramagnetic Mn. Another two of the close ( approximately 2.7 A) hydrogen nuclei show no isotropic couplings and are therefore most probably not contained in Mn ligands. We suggest that these proximal hydrogens may be associated with a Ca(2+)-bound substrate, as indicated in recent mechanistic proposals for O(2) formation.

Field-swept pulsed electron paramagnetic resonance ofCr3+-doped ZBLAN fluoride glass

Field-swept pulsed electron paramagnetic resonance (EPR) spectraof a ZBLAN fluoride glass doped with a low concentration of Cr3+ areobtained using echo-detected EPR and hole-burning free induction decaydetection. We review the utility of the pulsed EPR technique in generatingfield-swept EPR spectra, as well as some of the distorting effects thatare peculiar to the pulsed detection method. The application of thistechnique to Cr3+-doped ZBLAN reveals that much of the broadresonance extending from geff = 5.1 to geff = 1.97,characteristic of X-band continuous wave EPR of Cr3+ in glasses, isabsent. We attribute this largely to the variation in nutation frequenciesacross the spectrum that result from sites possessing large fine structureinteractions. The description of the spin dynamics of such sites iscomplicated and we discuss some possible approaches to the simulation ofthe pulsed EPR spectra.

EPR/ENDOR characterization of the physical and electronic structure of the OEC Mn cluster

Electron paramagnetic resonance (EPR) spectroscopy has often played a crucial role in characterizing the various cofactors and processes of photosynthesis, and photosystem II and its oxygen evolving chemistry is no exception. Until recently, the application of EPR spectroscopy to the characterization of the oxygen evolving complex (OEC) has been limited to the S 2-state of the Kok cycle. However, in the past few years, continuous wave-EPR signals have been obtained for both the S 0- and S 1-state as well as for the S 2–Y Z-state of a number of inhibited systems. Furthermore, the pulsed EPR technique of electron spin echo electron nuclear double resonance spectroscopy has been used to directly probe the 55Mn nuclei of the manganese cluster. In this review, we discuss how the EPR data obtained from each of these states of the OEC Kok cycle are being used to provide insight into the physical and electronic structure of the manganese cluster and its interaction with the key tyrosine, Y Z.

Uptake of Cu 2+ by the calcium carbonates vaterite and calcite as studied by continuous wave (cw) and pulse electron paramagnetic resonance

The investigation of the Cu 2+ uptake by the calcium carbonate minerals vaterite and calcite with continuous wave and pulse electron paramagnetic resonance (EPR) yields information on a molecular scale about the relevant complexation reactions at the mineral–water interface. The structural assignment is based on changes in the coordination geometry of the copper complexes. Magnetic interactions of the unpaired Cu 2+ electron with nuclei of 13C-labeled carbonate ligands and protons from water or hydroxyl ligands in the first and second coordination spheres of the cation are detected by pulse EPR techniques. Our results show that the Cu 2+ ions are rapidly dehydrated upon adsorption on the mineral surface. The strong surface binding is due to monodentate coordination to three or four carbonate surface ions, comparable to chelate complexation in solution. The formation of square-planar or square-pyramidal copper complexes at exposed surface sites like kinks and steps yields a convincing explanation for the inhibition of calcium carbonate growth and dissolution. Upon recrystallization the Cu 2+ ions are integrated into the calcite lattice where they exhibit a dynamic Jahn–Teller effect. The resulting local lattice distortions are expected to destabilize the Cu x Ca (1− x) CO 3( s) solid solution. Our results support the concept of a dynamic calcium carbonate surface, covered by a thin, structured surface layer. The detailed structural information obtained for Cu 2+ provides a better understanding of the interaction of other metal ions with calcium carbonate minerals.

Temperature-dependent pulsed electron paramagnetic resonance studies of the S2 state multiline signal of the photosynthetic oxygen-evolving complex.

The electron spin-lattice relaxation rate (1/T1) of the g = 2 "multiline" manganese electron paramagnetic resonance (EPR) signal arising from the photosystem II oxygen-evolving complex poised in the S2 state has been directly measured over the temperature range of 4.2-11 K via the inversion-recovery pulsed EPR technique. The electron spin echo amplitude of the g = 2 "multiline" signal varies inversely with temperature over this range, indicating a ground spin state Curie law behavior in agreement with our previously reported work [Britt et al. (1992) Biochim. Biophys. Acta 1140, 95-101]. Results of a plot of the natural log of the electron spin-lattice relaxation rate versus reciprocal temperature are consistent with an Orbach mechanism serving as the dominant relaxation pathway for the "multiline" signal in this temperature range. The slope of the plot indicates that an excited spin state manifold exists 36.5 cm-1 above the ground-state manifold that gives rise to the "multiline" signal.

Probing molecular cages in polymeric gels using paramagnetic ions: Internal motion of cupric ion in a cage

The effect of high-energy irradiation on the aqueous solutions of certain water soluble polymers, namely, poly(vinyl alcohol) and poly(ethylene oxide), brings about physical and chemical changes. The cross links or networks formed in the polymer chains cause gel formation. These gels are considered to be made up of cubic cells or molecular cages with size varying between 0.6 and 1.5 nm. Such cages have been recently probed using the paramagnetic ions of hydrated cupric as well as vanadyl ions and were studied by electron paramagnetic resonance (EPR) and pulsed EPR techniques. The time-dependent EPR spectra of Cu2+ ions in the cages are analyzed in this study using specific motional models, employing the modified Bloch equations. Computer simulations show that the spin probe’s motion is governed by the segmental motion of the polymer chains.