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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., Magnetochemistry;2018, 4(4), 50

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 Lechosław Latos-Grażyński 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.

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.

International audience; In the last decade, joint efforts of biologists, chemists and physicists have helped in understanding the dominant factors determining specificity and directionality of transmembrane transfer processes in proteins. In this endeavor, electron paramagnetic resonance (EPR) spectroscopy has played an important role. Characteristic examples of such determining factors are hydrogen-bonding patterns and polarity effects of the microenvironment of protein sites involved in the transfer process. These factors may undergo characteristic changes during the reaction and, thereby, control the efficiency of biological processes, e.g. light-induced electron and proton transfer across photosynthetic membranes or ion-channel formation of bacterial toxins. In case the transfer process does not involve stable or transient paramagnetic species or states, site-directed spin labeling with suitable nitroxide radicals still allows EPR techniques to be used for studying structure and conformational dynamics of the proteins in action. By combining site-directed spin labeling with high-field/high-frequency EPR, unique information on the proteins is revealed, which is complementary to that of X-ray crystallography, solid-state NMR, FRET, fast infrared and optical spectroscopic techniques. The main object of this publication is twofold: (i) to review our recent spin-label high-field EPR work on the bacteriorhodopsin light-driven proton pump from Halobacterium salinarium and the Colicin A ion-channel forming bacterial toxin produced in Escherichia coli, (ii) to report on novel high-field EPR experiments for probing site-specific pK(a) values in protein systems by means of pH-sensitive nitroxide spin labels. Taking advantage of the improved spectral and temporal resolution of high-field EPR at 95 GHz/3.4 T and 360 GHz/12.9 T, as compared to conventional X-band EPR (9.5 GHz/0.34 T), detailed information on the transient intermediates of the proteins in biological action is obtained. These intermediates can be observed and characterized while staying in their working states on biologically relevant timescales. The paper concludes with an outlook of ongoing high-field EPR experiments on site-specific protein mutants in our laboratories at FU Berlin and Osnabrück.

The light-induced electron transfer reaction of flavin cofactor photoactivation in Xenopus laevis (6–4) photolyase has been studied by continuous-wave and time-resolved electron paramagnetic resonance spectroscopy. When the photoactivation is initiated from the fully oxidized form of the flavin, a neutral flavin radical is observed as a long-lived paramagnetic intermediate of two consecutive single-electron reductions under participation of redox-active amino acid residues. By time-resolved electron paramagnetic resonance, a spin-polarized transient radical-pair signal was detected that shows remarkable differences to the signals observed in the related cyclobutane pyrimidine dimer photolyase enzyme. In (6–4) photolyase, a neutral tyrosine radical has been identified as the final electron donor, on the basis of the characteristic line width, hyperfine splitting pattern, and resonance magnetic field position of the tyrosine resonances of the transient radical pair.

Pulsed high-field/high-frequency EPR (3.4 T magnetic field, 95 GHz microwave frequency) is performed on the primary electron acceptor ubisemiquinone-10 anion radical (UQ-10 −. ) in Zn-substituted photosynthetic reaction centers (RCs) of Rhodobacter sphaeroides R26. In addition, in vitro measurements of UQ-10 −. in a frozen solution of deuterated isopropanol are presented. The method is two-dimensional EPR, with the swept magnetic field as the first and the pulse separation time as the second variable of the echo-detected spectra. The high Zeeman field leads to a spectral resolution of the anisotropic g -tensor. This allows a sensitive detection of the orientation-dependent echo-decay functions. The T 2 relaxation anisotropy at high field is dominated by librations. Comparison of in vivo and in vitro measurements shows different anisotropies of the echo-decay functions. The results are interpreted by calculations considering angular motions around the principal axes of the g -tensor. Hydrogen bonds between UQ-10 −. and its protic environment are considered.

A time-resolved ENDOR spectrum of the triplet state of the primary donor of photosystem II in isolated reaction centers has been obtained at cryogenic temperatures. Selective excitation of donor molecules, which have their z triplet axes parallel to the magnetic field, has permitted the measurement of the A zz component of the hyperfine coupling tensor of protons, in the reference frame of the zero field splitting tensor. For comparison, measurements have also been made on the photoexcited triplet state of monomeric chlorophyll a in vitro. The results indicate that in the temperature range 10–20 K the triplet state of the primary donor of photosystem II is largely located on a monomeric chlorophyll a molecule.

This study aims to investigate the g xx heterogeneity of the g-tensor commonly observed in high-field electron paramagnetic resonance (EPR) spectra of nitroxide spin-labeled sites in proteins. This heterogeneity is addressed in terms of spin-label populations characterized by different polarity and H-bonding properties of the nitroxide micro-environment. The g xx value for each population is determined from the fit of continuous-wave high-field spectra obtained at 95, 275 and 360 GHz with a series of nitroxide spin-labels covalently attached to different sites in both membrane and water-soluble proteins. The spin-labeled proteins investigated include sensory rhodopsin II and its cognate transducer molecule (HtrII) from Natronomonas pharaonis both in micelles and membranes, bacteriorhodopsin from Halobacterium salinarum in native purple membrane lipid bilayers and water-soluble colicin A from Escherichia coli. To avoid contributions to the g xx spectral features of the nitroxide label due to nuclear quadrupole interactions arising from 14N nuclei, and to simplify the nitrogen hyperfine pattern, methanethiosulfonate spin labels, containing the 15N isotope (I = 1/2) in some experiments, were employed. A consistent analysis of all multi-frequency EPR spectra revealed three distinct g xx values, g , for each investigated position of the labeled proteins. In contrast, distinctly different nitrogen hyperfine splittings A zz of the nitroxides in the various labeled proteins could not be resolved, but rather an average hyperfine splitting $$\bar{A}_{{zz}}$$ was obtained. The g values as well as the fractions of the different nitroxide populations were found to be correlated with the average hyperfine constant $$\bar{A}_{{zz}},$$ a parameter which likewise is known to be sensitive to the local polarity of the spin-label micro-environment. Plotting the different g values obtained for each EPR spectrum versus $$\bar{A}_{{zz}}$$ of the labeled proteins reveals new interesting aspects of the nitroxide label micro-environment in terms of polarity and H-bonding propensity (proticity). Linear approximations of the different regions of the plot g versus $$\bar{A}_{{zz}}$$ are presented and compared with theoretical and experimental data available from the literature.

Experiments have been performed on a model guest molecule, phenazine, in biphenyl single crystal and n-heptane polycrystalline host matrices to illustrate the two main contributions to inhomogeneous line broadening in ODMR spectroscopy. These arise from (a) inhomogeneous crystal fields experienced by the guest molecule in a host lattice and (b) unresolved hyperfine splitting. It is shown that the former may be reduced by making optical site selection during both excitation and detection. In the latter category, the most offensive group of nuclei is shown to be the guest protons and to a lesser extent the near-lying host protons. This contribution may be drastically reduced by fully deuterating both guest and host systems. Performing ODMR spectroscopy under these conditions allows the typical linewidths of 1–10 MHz to be reduced to ca. 200 kHz. The advantages of such high-resolution spectroscopy are manifold, not the least of which being that it forms a prerequisite for highly specific multiresonance experiments such as the recently developed ODNQR method.