High-field/high-frequency EPR spectroscopy in protein research: principles and examples

During the last decades, the combined efforts of biologists, chemists, and physicists in developing high-field/high-frequency EPR techniques and applying them to functional proteins have demonstrated that this type of magnetic resonance spectroscopy is particularly powerful for characterizing the structure and dynamics of stable and transient states of proteins in action on biologically relevant time scales ranging from nanoseconds to hours. The review article describes how high-field EPR methodology, in conjunction with site-specific isotope and spin-labeling strategies, is capable of providing new insights into fundamental biological processes. Specifically, we discuss the theoretical and instrumental background of continuous-wave and pulse high-field EPR and the multiple-resonance extensions EDNMR, ENDOR, TRIPLE, ESEEM, PELDOR, and RIDME. Some emphasis is placed on a balanced description of both the historical spadework and the achieved performance of advanced EPR at 95 GHz and 360 GHz. This culminates in a coherent treatment of state-of-the-art research of high-field EPR in terms of both instrumentation development and application to representative protein complexes such as cofactor binding sites in photosynthesis

High-field/High-frequency EPR Spectroscopy in Protein Research: Principles and Examples

During the last decades, the combined efforts of biologists, chemists, and physicists in developing high-field/high-frequency EPR techniques and applying them to functional proteins have demonstrated that this type of magnetic resonance spectroscopy is particularly powerful for characterizing the structure and dynamics of stable and transient states of proteins in action on biologically relevant time scales ranging from nanoseconds to hours. The review article describes how high-field EPR methodology, in conjunction with site-specific isotope and spin-labeling strategies, is capable of providing new insights into fundamental biological processes. Specifically, we discuss the theoretical and instrumental background of continuous-wave and pulse high-field EPR and the multiple-resonance extensions EDNMR, ENDOR, TRIPLE, ESEEM, PELDOR, and RIDME. Some emphasis is placed on a balanced description of both the historical spadework and the achieved performance of advanced EPR at 95 GHz and 360 GHz. This culminates in a coherent treatment of state-of-the-art research of high-field EPR in terms of both instrumentation development and application to representative protein complexes such as cofactor binding sites in photosynthesis

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

Möbius-Strip Topology of Expanded Porphyrins: A Minireview on EPR, ENDOR and DFT MO Studies

The one-sided Möbius strip with its characteristic 180° twist in the loop has inspired philosophers, artists and scientists since hundreds of years and continues to do so. On the molecular level, only in the last 15 years have some groups succeeded in synthesizing new expanded porphyrin compounds large enough to adopt Möbius-strip topology and Möbius aromaticity, the first being Lechosław Latos-Grażyński and collaborators in Wroclaw (2007) and Atsuhiro Osuka and collaborators in Kyoto (2008). We report on new studies of expanded porphyrins with either Möbius topology or Hückel topology that were synthesized in these laboratories. In this minireview, we focus on recent continuous-wave and time-resolved EPR, ENDOR and DFT MO studies on open-shell states of di-p-benzi[28]hexaphyrin(1.1.1.1.1.1), specifically, on the ground-state radical cation doublet state (total electron spin S = 1/2) and the first excited triplet state (S = 1). The review is largely based on the results and discussions of two previous publications: Möbius et al. (Appl Magn Reson 47:757–780, 2016) and Ema et al. (J Phys Chem Lett 9:2685–2690, 2018). In the open-shell systems, besides the electron-nuclear hyperfine couplings also the zero-field interaction tensor turned out to be a viable sensor for electronic structure changes between Möbius and Hückel topologies. In the Outlook section, we address the cyclotides, a new class of natural circular mini-proteins, usually less than 100 amino acids long. They are distinguished by exceptional chemical and biological stability. This is due to topological constraints imposed by threefoil knot and Möbius-strip formation. As a result, their physical qualities are “topologically protected”. This makes them highly interesting for medical or agricultural applications, for example as novel active ingredients against autoimmune diseases, viral infections, or as agents against insect damage to crops

Biomolecular EPR Meets NMR at High Magnetic Fields

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. View Full-Tex

Dielectric coupler for general purpose Q-band EPR cavity

Here, we report on a robust and efficient mechanism for tuning the microwave coupling of a Q-band (34 GHz), general purpose, cylindrical EPR cavity operating in the TE011 mode. This novel mechanism allows for both the adjustment of the cavity’s coupling over a wide frequency range, as well as its bandwidth from that of a high-Q cavity (about 10 MHz), to a broadband cavity (above 1 GHz). The coupling element consists of a dielectric plate fixed onto a movable waveguide short that allows for two modes of operation. In the first mode, the dielectric plate does not influence the resonance properties of the coupling iris and allows for precise, critical coupling of the high-Q cavity. In the second mode, the dielectric plate is positioned in front of the coupling iris, varying the iris’ resonance properties and allowing very strong overcoupling to be achieved. This mechanism can be generalized for other types of EPR cavities, in particular at high microwave frequencies

Hydrogen-Bonded Complexes of Neutral Nitroxide Radicals with 2-Propanol Studied by Multifrequency EPR/ENDOR

The hydrogen bond plays a key role in weak directional intermolecular interactions. It is operative in determining molecular conformation and aggregation, and controls the function of many chemical systems, ranging from inorganic, organic to biological molecules. Although an enormous amount of spectroscopic information has been collected about hydrogen-bond formation between molecules with closed-shell electronic configuration, the details of such interactions between open-shell radicals and closed-shell molecules are still rare. Here we report on an investigation of hydrogen-bonded complexes between pyrroline-type as well as piperidine-type neutral nitroxide radicals and an alcohol, i.e., 2-propanol. These nitroxide radicals are commonly used as EPR spin labels and probes. To obtain information on the geometry of the complexes and their electronic structure, multi-resonance EPR techniques at various microwave frequencies (X-, Q-, W-band, 244 GHz) have been employed in conjunction with DFT calculations. The planar five-membered ring system of the pyrroline-type nitroxide radical was found to form exclusively well-defined in-plane σ-type hydrogen-bonded complexes with one 2-propanol molecule in the first solvation shell in frozen solution. The measured hyperfine parameters of the hydrogen-bridge proton and the internal magnetic parameters describing the electron Zeeman and the electron-nuclear hyperfine and nuclear quadrupole interactions are in good agreement with values predicted by state-of-the-art DFT calculations. In contrast, multi-resonance EPR on the non-planar six-membered ring system of the piperidine-type nitroxide radical (TEMPOL) reveals a more complex situation, i.e., a mixture of a σ-type with, presumably, an out-of-plane π-type complex, both present in comparable fraction in frozen solution. For TEMPOL, the DFT calculations failed to predict magnetic interaction parameters that are in good agreement with experiment, apparently due to the considerable flexibility of the nitroxide and hydrogen-bonded complex. The detailed information about nitroxide/solvent complexes is of particular importance for Dynamic Nuclear Polarization (DNP) and site-directed spin-labeling EPR studies that employ nitroxides as polarizing agents or spin labels, respectively

Effect of Dehydrated Trehalose Matrix on the Kinetics of Forward Electron Transfer Reactions in Photosystem I

The effect of dehydration on the kinetics of forward electron transfer (ET) has been studied in cyanobacterial photosystem I (PS I) complexes in a trehalose glassy matrix by time-resolved optical and EPR spectroscopies in the 100 fs to 1 ms time domain. The kinetics of the flash-induced absorption changes in the subnanosecond time domain due to primary and secondary charge separation steps were monitored by pump–probe laser spectroscopy with 20-fs low-energy pump pulses centered at 720 nm. The back-reaction kinetics of P700 were measured by high-field time-resolved EPR spectroscopy and the forward kinetics of A∙−1A/A∙−1B→FX by time-resolved optical spectroscopy at 480 nm. The kinetics of the primary ET reactions to form the primary P∙+700A∙−0 and the secondary P∙+700A∙−1 ion radical pairs were not affected by dehydration in the trehalose matrix, while the yield of the P∙+700A∙−1 was decreased by ~20%. Forward ET from the phylloquinone molecules in the A∙−1A and A∙−1B sites to the iron–sulfur cluster FX slowed from ~220 ns and ~20 ns in solution to ~13 μs and ~80 ns, respectively. However, as shown by EPR spectroscopy, the ~15 μs kinetic phase also contains a small contribution from the recombination between A∙−1B and P∙+700. These data reveal that the initial ET reactions from P700 to secondary phylloquinone acceptors in the A- and B-branches of cofactors (A1A and A1B) remain unaffected whereas ET beyond A1A and A1B is slowed or prevented by constrained protein dynamics due to the dry trehalose glass matrix

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 Image ID:c7cp03942e-t1.gif (primary electron donor) and Image ID:c7cp03942e-t2.gif (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

Hydrogen-bonded complexes of neutral nitroxide radicals with 2-propanol studied by multifrequency EPR/ENDOR

The hydrogen bond plays a key role in weak directional intermolecular interactions. It is operative in determining molecular conformation and aggregation, and controls the function of many chemical systems, ranging from inorganic, organic to biological molecules. Although an enormous amount of spectroscopic information has been collected about hydrogen-bond formation between molecules with closed-shell electronic configuration, the details of such interactions between open-shell radicals and closed-shell molecules are still rare. Here we report on an investigation of hydrogen-bonded complexes between pyrroline-type as well as piperidine-type neutral nitroxide radicals and an alcohol, i.e., 2-propanol. These nitroxide radicals are commonly used as EPR spin labels and probes. To obtain information on the geometry of the complexes and their electronic structure, multi-resonance EPR techniques at various microwave frequencies (X-, Q-, W-band, 244 GHz) have been employed in conjunction with DFT calculations. The planar five-membered ring system of the pyrroline-type nitroxide radical was found to form exclusively well-defined in-plane σ-type hydrogen-bonded complexes with one 2-propanol molecule in the first solvation shell in frozen solution. The measured hyperfine parameters of the hydrogen-bridge proton and the internal magnetic parameters describing the electron Zeeman and the electron-nuclear hyperfine and nuclear quadrupole interactions are in good agreement with values predicted by state-of-the-art DFT calculations. In contrast, multi-resonance EPR on the non-planar six-membered ring system of the piperidine-type nitroxide radical (TEMPOL) reveals a more complex situation, i.e., a mixture of a σ-type with, presumably, an out-of-plane π-type complex, both present in comparable fraction in frozen solution. For TEMPOL, the DFT calculations failed to predict magnetic interaction parameters that are in good agreement with experiment, apparently due to the considerable flexibility of the nitroxide and hydrogen-bonded complex. The detailed information about nitroxide/solvent complexes is of particular importance for Dynamic Nuclear Polarization (DNP) and site-directed spin-labeling EPR studies that employ nitroxides as polarizing agents or spin labels, respectively