Jim Hyde and the ENDOR Connection: A Personal Account.

In this minireview, we report on our year-long EPR work, such as electron-nuclear double resonance (ENDOR), pulse electron double resonance (PELDOR) and ELDOR-detected NMR (EDNMR) at X-band and W-band microwave frequencies and magnetic fields. This report is dedicated to James S. Hyde and honors his pioneering contributions to the measurement of spin interactions in large (bio)molecules. From these interactions, detailed information is revealed on structure and dynamics of macromolecules embedded in liquid-solution or solid-state environments. New developments in pulsed microwave and sweepable cryomagnet technology as well as ultra-fast electronics for signal data handling and processing have pushed the limits of EPR spectroscopy and its multi-frequency extensions to new horizons concerning sensitivity of detection, selectivity of molecular interactions and time resolution. Among the most important advances is the upgrading of EPR to high magnetic fields, very much in analogy to what happened in NMR. The ongoing progress in EPR spectroscopy is exemplified by reviewing various multi-frequency electron-nuclear double-resonance experiments on organic radicals, light-generated donor-acceptor radical pairs in photosynthesis, and site-specifically nitroxide spin-labeled bacteriorhodopsin, the light-driven proton pump, as well as EDNMR and ENDOR on nitroxides. Signal and resolution enhancements are particularly spectacular for ENDOR, EDNMR and PELDOR on frozen-solution samples at high Zeeman fields. They provide orientation selection for disordered samples approaching single-crystal resolution at canonical g-tensor orientations-even for molecules with small g-anisotropies. Dramatic improvements of EPR detection sensitivity could be achieved, even for short-lived paramagnetic reaction intermediates. Thus, unique structural and dynamic information is revealed that can hardly be obtained by other analytical techniques. Micromolar concentrations of sample molecules have become sufficient to characterize stable and transient reaction intermediates of complex molecular systems-offering exciting applications for physicists, chemists, biochemists and molecular biologists.

Local water sensing: water exchange in bacterial photosynthetic reaction centers embedded in a trehalose glass studied using multiresonance EPR.

Using isotope labeled water (D2O and H217O) and pulsed W-band (94 GHz) high-field multiresonance EPR spectroscopies, such as ELDOR-detected NMR and ENDOR, the biologically important question of detection and quantification of local water in proteins is addressed. A bacterial reaction center (bRC) from Rhodobacter sphaeroides R26 embedded into a trehalose glass matrix is used as a model system. The bRC hosts the two native radical cofactor ions (primary electron donor) and (primary electron acceptor) as well as an artificial nitroxide spin label site-specifically attached to the surface of the H-protein domain. The three paramagnetic reporter groups have distinctly different local environments. They serve as local probes to detect water molecules via magnetic interactions (electron-nuclear hyperfine and quadrupole) with either deuterons or 17O nuclei. bRCs were equilibrated in an atmosphere of different relative humidities allowing us to control precisely the hydration levels of the protein. We show that by using oxygen-17 labeled water quantitative conclusions can be made in contrast to using D2O which suffers from proton-deuterium exchange processes in the protein. From the experiments we also conclude that dry trehalose operates as an anhydrobiotic protein stabilizer in line with the "anchorage hypothesis" of bio-protection. It predicts selective changes in the first solvation shell of the protein upon trehalose-matrix dehydration with subsequent changes in the hydrogen-bonding network. Changes in hydrogen-bonding patterns usually have an impact on the global function of a biological system.

Protein Immobilization Capabilities of Sucrose and Trehalose Glasses: The Effect of Protein/Sugar Concentration Unraveled by High-Field EPR.

Disaccharide glasses are increasingly used to immobilize proteins at room temperature for structural/functional studies and long-term preservation. To unravel the molecular basis of protein immobilization, we studied the effect of sugar/protein concentration ratios in trehalose or sucrose matrixes, in which the bacterial photosynthetic reaction center (RC) was embedded as a model protein. The structural, dynamical, and H-bonding characteristics of the sugar-protein systems were probed by high-field W-band EPR of a matrix-dissolved nitroxide radical. We discovered that RC immobilization and thermal stabilization, being independent of the protein concentration in trehalose, occur in sucrose only at sufficiently low sugar/protein ratios. EPR reveals that only under such conditions does sucrose form a microscopically homogeneous matrix that immobilizes, via H-bonds, the nitroxide probe. We conclude that the protein immobilization capability depends critically on the propensity of the glass-forming sugar to create intermolecular H-bond networks, thus establishing long-range, homogeneous connectivity within the matrix.

Möbius-Hückel topology switching in an expanded porphyrin cation radical as studied by EPR and ENDOR spectroscopy.

The symmetry of the arrangement of objects has fascinated philosophers, artists and scientists for a long time, and still does. Symmetries often exist in nature, but are also created artificially, for instance by chemical synthesis of novel molecules and materials. The one-sided, non-orientable Möbius band topology is a paradigm of such a symmetry-based fascination. In the early 1960s, in synthetic organic chemistry the interest in molecules with Möbius symmetry was greatly stimulated by a short paper by Edgar Heilbronner. He predicted that sufficiently large [n]annulenes with a closed-shell electron configuration of 4n π-electrons should allow for sufficient π-overlap stabilization to be synthesizable by twisting them with a 180° phase change into the Möbius symmetry of their hydrocarbon skeleton. In 2007, the group of Lechoslaw Latos-Grazynski succeeded in synthesizing the compound di-p-benzi[28]hexa-phyrin(1.1.1.1.1.1), compound 1, which can dynamically switch between Hückel and Möbius conjugation depending, in a complex manner, on the polarity and temperature of the surrounding solvent. This discovery of "topology switching" between the two-sided (Hückel) and one-sided (Möbius) molecular state with closed-shell electronic configuration was based primarily on the results of NMR spectroscopy and DFT calculations. The present EPR and ENDOR work on the radical cation state of compound 1 is the first study of a ground-state open-shell system which exhibits a Hückel-Möbius topology switch that is controlled by temperature, like in the case of the closed-shell precursor. The unpaired electron interacting with magnetic nuclei in the molecule is used as a sensitive probe for the electronic structure and its symmetry properties. For a Hückel conformer with its higher symmetry, we expect - and observe - fewer ENDOR lines than for a Möbius conformer. The ENDOR results are supplemented by and in accordance with theoretical calculations based on density functional theory at the ORCA level.

High-field ELDOR-detected NMR study of a nitroxide radical in disordered solids: towards characterization of heterogeneity of microenvironments in spin-labeled systems.

The combination of high-field EPR with site-directed spin-labeling (SDSL) techniques employing nitroxide radicals has turned out to be particularly powerful in probing the polarity and proticity characteristics of protein/matrix systems. This information is concluded from the principal components of the nitroxide Zeeman (g), nitrogen hyperfine (A) and quadrupole (P) tensors of the spin labels attached to specific sites. Recent multi-frequency high-field EPR studies underlined the complexity of the problem to treat the nitroxide microenvironment in proteins adequately due to inherent heterogeneities which result in several principal x-components of the nitroxide g-tensor. Concomitant, but distinctly different nitrogen hyperfine components could, however, not be determined from high-field cw EPR experiments owing to the large intrinsic EPR linewidth in fully protonated guest/host systems. It is shown in this work that, using the W-band (95GHz) ELDOR- (electron-electron double resonance) detected NMR (EDNMR) method, different principal nitrogen hyperfine, Azz, and quadrupole, Pzz, tensor values of a nitroxide radical in glassy 2-propanol matrix can be measured with high accuracy. They belong to nitroxides with different hydrogen-bond situations. The satisfactory resolution and superior sensitivity of EDNMR as compared to the standard ENDOR (electron-nuclear double resonance) method are demonstrated.

High-field EPR on membrane proteins - crossing the gap to NMR.

In this review on advanced EPR spectroscopy, which addresses both the EPR and NMR communities, considerable emphasis is put on delineating the complementarity of NMR and EPR concerning the measurement of molecular interactions in large biomolecules. From these interactions, detailed information can be revealed on structure and dynamics of macromolecules embedded in solution- or solid-state environments. New developments in pulsed microwave and sweepable cryomagnet technology as well as ultrafast electronics for signal data handling and processing have pushed to new horizons the limits of EPR spectroscopy and its multifrequency extensions concerning the sensitivity of detection, the selectivity with respect to interactions, and the resolution in frequency and time domains. One of the most important advances has been the extension of EPR to high magnetic fields and microwave frequencies, very much in analogy to what happens in NMR. This is exemplified by referring to ongoing efforts for signal enhancement in both NMR and EPR double-resonance techniques by exploiting dynamic nuclear or electron spin polarization via unpaired electron spins and their electron-nuclear or electron-electron interactions. Signal and resolution enhancements are particularly spectacular for double-resonance techniques such as ENDOR and PELDOR at high magnetic fields. They provide greatly improved orientational selection for disordered samples that approaches single-crystal resolution at canonical g-tensor orientations - even for molecules with small g-anisotropies. Exchange of experience between the EPR and NMR communities allows for handling polarization and resolution improvement strategies in an optimal manner. Consequently, a dramatic improvement of EPR detection sensitivity could be achieved, even for short-lived paramagnetic reaction intermediates. Unique structural and dynamic information is thus revealed that can hardly be obtained by any other analytical techniques. Micromolar quantities of sample molecules have become sufficient to characterize stable and transient reaction intermediates of complex molecular systems - offering highly interesting applications for chemists, biochemists and molecular biologists. In three case studies, representative examples of advanced EPR spectroscopy are reviewed: (I) High-field PELDOR and ENDOR structure determination of cation-anion radical pairs in reaction centers from photosynthetic purple bacteria and cyanobacteria (Photosystem I); (II) High-field ENDOR and ELDOR-detected NMR spectroscopy on the oxygen-evolving complex of Photosystem II; and (III) High-field electron dipolar spectroscopy on nitroxide spin-labelled bacteriorhodopsin for structure-function studies. An extended conclusion with an outlook to further developments and applications is also presented.

Incorporation of a high potential quinone reveals that electron transfer in Photosystem I becomes highly asymmetric at low temperature.

Photosystem I (PS I) has two nearly identical branches of electron-transfer co-factors. Based on point mutation studies, there is general agreement that both branches are active at ambient temperature but that the majority of electron-transfer events occur in the A-branch. At low temperature, reversible electron transfer between P(700) and A(1A) occurs in the A-branch. However, it has been postulated that irreversible electron transfer from P(700) through A(1B) to the terminal iron-sulfur clusters F(A) and F(B) occurs via the B-branch. Thus, to study the directionality of electron transfer at low temperature, electron transfer to the iron-sulfur clusters must be blocked. Because the geometries of the donor-acceptor radical pairs formed by electron transfer in the A- and B-branch differ, they have different spin-polarized EPR spectra and echo-modulation decay curves. Hence, time-resolved, multiple-frequency EPR spectroscopy, both in the direct-detection and pulse mode, can be used to probe the use of the two branches if electron transfer to the iron-sulfur clusters is blocked. Here, we use the PS I variant from the menB deletion mutant strain of Synechocyctis sp. PCC 6803, which is unable to synthesize phylloquinone, to incorporate 2,3-dichloro-1,4-naphthoquinone (Cl(2)NQ) into the A(1A) and A(1B) binding sites. The reduction midpoint potential of Cl(2)NQ is approximately 400 mV more positive than that of phylloquinone and is unable to transfer electrons to the iron-sulfur clusters. In contrast to previous studies, in which the iron-sulfur clusters were chemically reduced and/or point mutations were used to prevent electron transfer past the quinones, we find no evidence for radical-pair formation in the B-branch. The implications of this result for the directionality of electron transfer in PS I are discussed.

High-field dipolar electron paramagnetic resonance (EPR) spectroscopy of nitroxide biradicals for determining three-dimensional structures of biomacromolecules in disordered solids.

We consider the state-of-the-art capabilities and future perspectives of electron-spin triangulation by high-field/high-frequency dipolar electron paramagnetic resonance (EPR) techniques designed for determining the three-dimensional structure of large supra-molecular complexes dissolved in disordered solids. These techniques combine double site-directed spin labeling (SDSL) with orientation-resolving pulsed electron-electron double resonance (PELDOR) spectroscopy. In particular, we appraise the prospects of angular triangulation, which extends the more familiar distance triangulation. As a model case for spin-labeled proteins, the three-dimensional structures of two nitroxide biradicals with rather stiff bridging blocks and deuterated nitroxide headgroups have been derived. To this end we applied 95 GHz high-field electron dipolar EPR spectroscopy with the microwave pulse-sequence configurations for PELDOR and relaxation-induced dipolar modulation enhancement (RIDME). Various specific spectroscopic strategies are discussed to overcome the problems of overlapping spectra of the chemically identical nitroxide labels when attached to macromolecular systems. We conclude that due to the high detection sensitivity and spectral resolution the combination of SDSL with high-field RIDME/PELDOR stands out as an extremely powerful tool for 3D structure determination of large disordered systems. The approach compares favorably with other structure-determining magnetic-resonance methods. This holds true both for stable and transient radical-pair states. Angular constraints are provided in addition to distance constraints obtained for the same sample. Thereby, the number of necessary distance constraints is strongly reduced. Since each measurement of a distance constraint requires an additional doubly spin-labeled sample, the reduction of necessary distance constraints is another appealing aspect of orientation-resolving EPR spin triangulation for protein structure determination.

Electron-nuclear and electron-electron double resonance spectroscopies show that the primary quinone acceptor QA in reaction centers from photosynthetic bacteria Rhodobacter sphaeroides remains in the same orientation upon light-induced reduction.

Reaction centers (RCs) from the photosynthetic bacterium Rhodobacter (Rb.) sphaeroides R-26 exhibit changes in the recombination kinetics of the charge-separated radical-pair state, P(·+) Q(A)(·-), composed of the dimeric bacteriochlorophyll donor P and the ubiquinone-10 acceptor Q(A), depending on whether the RCs are cooled to cryogenic temperatures in the dark or under continuous illumination (Kleinfeld et al. Biochemistry 1984, 23, 5780-5786). Structural changes near redox-active cofactors have been postulated to be responsible for these changes in kinetics and to occur in the course of light-induced oxidation and reduction of the cofactors thereby assuring a high quantum yield. Here we investigated such potential light-induced structural changes, associated with the formation of P(·+) Q(A)(·-), via pulsed electron-nuclear double resonance (ENDOR) at Q-band (34 GHz) and pulsed electron-electron double resonance (PELDOR) at W-band (95 GHz). Two types of light excitation have been employed for which identical RC samples were prepared: (a) one sample was frozen in the dark and then illuminated to generate transient P(·+) Q(A)(·-), and (b) one was frozen under illumination which resulted in both trapped and transient P(·+) Q(A)(·-) at 80 K. The hyperfine interactions between Q(A)(·-) and the protein were found to be the same in RCs frozen in the dark as in RCs frozen under illumination. Furthermore, these interactions are completely consistent with those observed in RC crystals frozen in the dark. Thus, QA remains in its binding site with the same position and orientation upon reduction. This conclusion is consistent with the result of our orientation-resolving PELDOR experiments on transient P(·+) Q(A)(·-) radical pairs. However, these findings are incompatible with the recently proposed ~60° reorientation of Q(A) upon its photoreduction, as deduced from an analysis of Q-band quantum-beat oscillations (Heinen et al. J. Am. Chem. Soc. 2007, 129, 15935-15946). Such a large reorientation appears improbable, and our objections against this proposition are substantiated here in detail. Our results show that Q(A) is initially in an orientation that is favorable for its light-driven reduction. This diminishes the reorganization requirements for fast electron reduction and high quantum efficiency.

Bacterial photosynthetic reaction centers in trehalose glasses: coupling between protein conformational dynamics and electron-transfer kinetics as studied by laser-flash and high-field EPR spectroscopies.

The coupling between electron transfer (ET) and the conformational dynamics of the cofactor−protein complex in photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides in water/glycerol solutions or embedded in dehydrated poly(vinyl alcohol) (PVA) films or trehalose glasses is reported. Matrix effects were studied by time-resolved 95 GHz high-field electron paramagnetic resonance (EPR) spectroscopy at room (290 K) and low (150 K) temperature. ET from the photoreduced quinone acceptor (QA•−) to the photo-oxidized donor (P865•+) is strongly matrix-dependent at room temperature: In the trehalose glasses, the recombination kinetics of P865•+QA•−, probed by EPR and optical spectroscopies, is faster and broadly distributed as compared to that of RCs in solution, reflecting the inhibition of the RC relaxation from the dark- to the light-adapted conformational substate and the hindrance of substate interconversion. Similarly accelerated kinetics was observed also in PVA at a water-to-RC molar ratio 10-fold lower than in trehalose. Despite the matrix dependence of the ET kinetics, continuous-wave (cw) EPR and electron spin echo (ESE) analyses of the photogenerated P865•+ and QA•− radical ions and P865•+QA•− radical pairs do not reveal significant matrix effects, at either 290 or 150 K, indicating no change in the molecular radical-pair configuration of the P865•+ and QA•− cofactors. Furthermore, the field dependences of the transverse relaxation times T2 of QA•− essentially coincide in trehalose and PVA at 290 K. T2 is similar in these two matrixes and in the glycerol/water system at 150 K, implying that the librational dynamics of QA•− are also unaffected by the matrix. We infer that the relative geometry of the primary donor and acceptor, as well as the local dynamics and hydrogen bonding of QA in its binding pocket, are not involved in the stabilization of P865•+QA•−. We suggest that the RC relaxation occurs rather by changes throughout the protein/solvent system. The control of the RC dynamics and ET by the environment is discussed, particularly with respect to the extraordinary efficacy of trehalose matrixes in restricting the RC motional degrees of freedom at elevated temperatures.

Multifrequency EPR study of the mobility of nitroxides in solid-state calixarene nanocapsules.

Multifrequency continuous wave (cw) and echo-detected (ED) electron paramagnetic resonance (EPR) was employed to study the mobility of nitroxides confined in nanocapsules. The complexes p-hexanoyl calix[4]arene with 4-methoxy-2,2,6,6-tetramethylpiperidine-N-oxyl (MT) and N-(2-methylpropyl)-N-(1-diethylphosphono-2,2-dimethylpropyl)-aminoxyl (DEPN) were studied by X-, W-band and 360 GHz cw EPR at temperatures between 90 and 370 K. Thereby we were able to extract the canonical values of the hyperfine and g-tensors of the encapsulated radicals as well as information on restricted orientational dynamics of the caged spin probes. Comparing cw and ED-EPR data obtained on MT@C6OH we found that between 90 and 200 K the caged nitroxide undergoes isotropic small-angle fluctuations (librations), whereas at higher temperatures restricted rotations of the radical with correlation times of 0.75 x 10(-10) s and 1.2 x 10(-10) s dominate at 325 and 300 K, respectively. The activation energy of the rotational motion of encapsulated MT radicals was evaluated as E(a) = 1.0 kcal mol(-1), which is comparable to the magnitude of van der Waals interactions. Compared to MT, the reorientational motion of DEPN was found to be slower and more isotropic.

High-field EPR.

Among the numerous spectroscopic techniques utilized in photosynthesis research, high-field/high-frequency EPR and its pulse extensions ESE, ENDOR, ESEEM, and PELDOR play an important role in the endeavor to understand, on the basis of structure and dynamics data, dominant factors that control specificity and efficiency of light-induced electron- and proton-transfer processes in primary photosynthesis. Short-lived transient intermediates of the photocycle can be characterized by high-field EPR techniques, and detailed structural information can be obtained even from disordered sample preparations. The chapter describes how multifrequency high-field EPR methodology, in conjunction with mutation strategies for site-specific isotope or spin labeling and with the support of modern quantum-chemical computation methods for data interpretation, is capable of providing new insights into the photosynthetic transfer processes. The information obtained is complementary to that of protein crystallography, solid-state NMR and laser spectroscopy.

Inclusion of 4-methoxy-2,2,6,6-tetramethylpiperidine-N-oxyl in a calixarene nanocapsule in the solid state.

We present an X-ray diffraction (XRD) and multi frequency electron spin resonance (ESR) study of the structure and dynamics of an inclusion complex of p-hexanoyl calix[4]arene (C6OH) with 4-methoxy-2,2,6,6-tetramethylpiperidine-N-oxyl (MT). The single crystal XRD experiments reveal that MT along with ethanol (solvent) molecules are entrapped in a capsular type crystalline lattice of the host C6OH material. ESR measurements were performed at 9.2 GHz/0.33 T (X-band) and at 360 GHz/14 T. In order to avoid ESR line broadening resulting from electron dipole-dipole interaction between nitroxides occupying neighbouring capsules in the crystal lattice, the capsules containing nitroxides were separated from each other by capsules containing diamagnetic dibenzylketone (DBK). Due to the extremely high g-tensor resolution of ESR at 360 GHz, we were able to distinguish, by shifts of their gxx component, between encapsulated nitroxide molecules forming a hydrogen bond between their O-(N) group and the OH group of an ethanol molecule occupying the same capsule and nitroxides missing this interaction. Temperature dependent ESR measurements revealed an orientational anisotropy in the motion of MT encapsulated in C6OH. Solid lipid nanoparticles (SLN) prepared from C6OH and loaded with the nitroxide retained the microcrystalline capsular structure of the pertinent inclusion complex. We found that encapsulated MT in SLNs becomes inaccessible to reducing agents such as sodium ascorbate.

G-tensors of the flavin adenine dinucleotide radicals in glucose oxidase: a comparative multifrequency electron paramagnetic resonance and electron-nuclear double resonance study.

The flavin adenine dinucleotide (FAD) cofactor of Aspergillus niger glucose oxidase (GO) in its anionic (FAD*-) and neutral (FADH*) radical form was investigated by electron paramagnetic resonance (EPR) at high microwave frequencies (93.9 and 360 GHz) and correspondingly high magnetic fields and by pulsed electron-nuclear double resonance (ENDOR) spectroscopy at 9.7 GHz. Because of the high spectral resolution of the frozen-solution continuous-wave EPR spectrum recorded at 360 GHz, the anisotropy of the g-tensor of FAD*- could be fully resolved. By least-squares fittings of spectral simulations to experimental data, the principal values of g have been established with high precision: gX=2.00429(3), gY=2.00389(3), gZ=2.00216(3) (X, Y, and Z are the principal axes of g) yielding giso=2.00345(3). The gY-component of FAD*- from GO is moderately shifted upon deprotonation of FADH*, rendering the g-tensor of FAD*- slightly more axially symmetric as compared to that of FADH*. In contrast, significantly altered proton hyperfine couplings were observed by ENDOR upon transforming the neutral FADH* radical into the anionic FAD*- radical by pH titration of GO. That the g-principal values of both protonation forms remain largely identical demonstrates the robustness of g against local changes in the electron-spin density distribution of flavins. Thus, in flavins, the g-tensor reflects more global changes in the electronic structure and, therefore, appears to be ideally suited to identify chemically different flavin radicals.

Molecular dynamics of nitroxides in glasses as studied by multi-frequency EPR.

Pulsed multi-frequency EPR was used to investigate orientational molecular motion of the nitroxide spin probe (Fremy's salt) in glycerol glass near the glass transition temperature. By measuring echo-detected EPR spectra at different pulse separation times at resonance frequencies of 3, 9.5, 95 and 180 GHz, we were able to discriminate between different relaxation mechanisms and characterize the timescale of molecular reorientations (10(-7)-10(-10) s). We found that near the glass transition temperature, the orientation-dependent transverse relaxation is dominated by fast reorientational fluctuations, which may be overlapped with fast modulations of the canonical g-matrix values. The data was interpreted using a new simulation program for the orientation-dependent transverse relaxation rate 1/T2 of nitroxides based on different models for the molecular motion. The validity of the different models was assessed by comparing least-square fits of the simulated relaxation behaviour to the experimental data.

Intramolecular electron and energy transfer in an axial ZnP-pyridylfullerene complex as studied by X- and W-band time-resolved EPR spectroscopy.

Light-driven electron transfer (ET) and energy transfer (EnT) in a self-assembled via axial coordination Zn-porphyrin-pyridylfullerene (ZnP-PyrF) complex were studied by time-resolved electron paramagnetic resonance (TREPR) spectroscopy at 9.5 GHz (X-band) and 95 GHz (W-band). The studies over a wide temperature range were carried out in media of different polarity, including isotropic toluene and tetrahydrofuran (THF), and anisotropic nematic liquid crystals (LCs), E-7 and ZLI-4389. At low temperatures (frozen matrices), photoexcitation of the ZnP donor results mainly in singlet-singlet EnT to the pyridine-appended fullerene acceptor. In fluid phases ET is the dominant process. Specifically, in isotropic solvents the generated radical pairs (RPs) are long-lived, with lifetimes exceeding that observed for covalently linked donor-acceptor systems. It is concluded that in liquid phases of both polar and nonpolar solvents the separation of the tightly bound complex into the more loosely bound structure slows down the back ET (BET) process. Photoexcitation of the donor in fluid phases of LCs does not result in the creation of the long-lived RPs, since the ordered LC matrix hinders the separation of the complex constituents. As a result, fast intramolecular BET takes place in the tightly bound complex. Contrarily to the behavior of covalently linked donor-acceptor systems in different LCs, the polarity of the LC matrix affects the ET process. Moreover, in contrast to covalently linked D-s-A systems, utilization of LCs for the coordinatively linked D-s-A complexes does not reduce the ET rates significantly.

Multifrequency time-resolved electron paramagnetic resonance investigations after photolysis of phosphine oxide photoinitiators. Dependence of triplet mechanism chemically induced dynamic electron polarization on microwave frequency.

Phosphinoyl radicals were produced in benzene solution by photolysis of three acylphosphine oxide photoinitiators, diphenyl-2,4,6-trimethylbenzoyl phosphine oxide (I), bis(2,6-dimethoxybenzoyl)-(2,4,4-trimethylpentyl) phosphine oxide (II), and bis(2,4,6-trimethylbenzoyl) phenylphospine oxide (III). The chemically induced dynamic electron polarization (CIDEP) of the radicals was measured by time-resolved electron paramagnetic resonance spectroscopy at different microwave frequencies/magnetic fields, in S- (2.8 GHz, 0.1 T), X- (9.7 GHz, 0.34 T), Q- (34.8 GHz, 1.2 T), and W-bands (95 GHz, 3.4 T). The CIDEP was found to be due to a triplet mechanism (TM) superimposed by a radical pair mechanism comprising ST(0) as well as ST(-) mixing. Contributions of the different CIDEP mechanisms were separated, and the dependence of the TM polarization on microwave frequency was determined. It agrees well with the numerical solution of the relevant stochastic Liouville equation, which proves the TM theory quantitatively. The applicability of previous approximate analytical formulas for the TM polarization is discussed. Parameters of the excited triplet state of III were estimated from the dependence of the TM polarization on microwave frequency. They are zero-field splitting constant 0.169 cm(-1)

Dynamics in the Mn2+ binding site in single crystals of concanavalin A revealed by high-field EPR spectroscopy.

EPR spectroscopy at 95 GHz was used to characterize the dynamics at the Mn(2+) binding site in single crystals of the saccharide-binding protein concanavalin A. The zero-field splitting (ZFS) tensor of the Mn(2+) was determined from rotation patterns in the a-c and a-b crystallographic planes, acquired at room temperature and 4.5 K. The analysis of the rotation patterns showed that while at room temperature there is only one type of Mn(2+) site, at low temperatures two types of Mn(2+) sites, not related by any symmetry, are distinguished. The sites differ in the ZFS parameters D and E and in the orientation of the ZFS tensor with respect to the crystallographic axes. Temperature-dependent EPR measurements on a crystal oriented with its crystallographic a axis parallel to the magnetic field showed that as the temperature increases, the two well-resolved Mn(2+) sextets gradually coalesce into a single sextet at room temperature. The line shape changes are characteristic of a two-site exchange. This was confirmed by simulations which gave rates in the range of 10(7)-10(8) s(-1) for the temperature range of 200-266 K and an activation energy of 23.8 kJ/mol. This dynamic process was attributed to a conformational equilibrium within the Mn(2+) binding site which freezes into two conformations at low temperatures.