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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.

In memory of the 85th birthday of Yakov S. Lebedev (Moscow), who died in 1996, we start this Review on soft-glass matrix effects in donor–acceptor complexes with an appreciation of his pioneering work on high-field EPR spectroscopy on tribochemically generated donor–acceptor complexes. The mechanochemical activation of polycrystalline mixtures of porphyrins (and other donors) and quinone acceptors was found to produce large concentrations of triplet donor molecules and donor–acceptor radical pairs with unusual stability. The Review is continued with reporting on W-band high-field EPR and fast-laser studies on disaccharide matrix effects on structure and dynamics of donor–acceptor protein complexes related to photosynthesis, including the non-oxygenic bacterial reaction center (RC) and the oxygenic RCs Photosystem I (PS I) and Photosystem II (PS II, preliminary results). Some organisms can survive complete dehydration and high temperatures by adopting an anhydrobiotic state in which the intracellular medium contains large amounts of disaccharides, in particular trehalose and sucrose. Trehalose is most effective in protecting biostructures, both in vivo and in vitro. To clarify the molecular mechanisms of disaccharide bioprotection, structure and dynamics of sucrose and trehalose matrices at different controlled hydration levels were probed by perdeuterated nitroxide spin labels and native cofactor intermediates in their charge-separated states. Trehalose forms a homogeneous amorphous phase in which the hosted molecules are uniformly distributed. Notably, their rotational mobility at room temperature is dramatically impaired by thetrehalose H-bonding network confinement to an extent that in normal protein–matrix systems is only observed at low temperatures around 150K. From the experimental results, formation of an extended H-bonding network of trehalose with protein molecules is inferred, involving both bulk and local water molecules. The H-bond network extends homogeneously over the whole matrix integrating and immobilizing the hosted protein. Taken together, these observations suggest that in photosystems, such as bacterial RCs and PS I complexes, of different size and complexity regarding subunit composition and oligomeric organization, the molecular configuration of the cofactors involved in the primary processes of charge separation is not significantly distorted by incorporation into trehalose glass, even under extensive dehydration. By means of pulsed W-band high-field multiresonance EPR spectroscopies, such as ELDOR-detected NMR and ENDOR, in conjunction with using isotope labeled water (D2O and H217O), the biologically important issue of sensing and quantification of local water in proteins is addressed. The bacterial RC embedded into the trehalose glass matrix is used as model system. The two native radical cofactor ions of the primary electron donor and acceptor as well as an artificial nitroxide spin label site-specifically attached to the protein surface are studied in the experiments. The three paramagnetic reporter groups probe distinctly different local environments. They sense water molecules via their magnetic hyperfine and quadrupole interactions with either deuterons or 17O nuclei. It is shown that by using oxygen-17 labeled water, quantitative conclusions can be drawn differentiating between local and bulk water. It is concluded that dry trehalose operates as anhydrobiotic protein stabilizer by means of selective changes in the first solvation shell of the protein upon trehalose–matrix dehydration with subsequent changes in the hydrogen-bonding network. Such changes usually have an impact on the global function of a biological system. Finally, preliminary results of optical and W-band EPR experiments on the extremolytes ectoine and its derivative hydroxyectoine are reported; these compounds appear to share several stress-protecting properties with trehalose in terms of stabilizing protein matrices. For instance, they display remarkable stabilizing capabilities towards sensitive proteins and enzymes with respect to freeze-thawing, heat-treatment, and freeze-drying procedures. Moreover, hydroxyectoine is a good glass-forming compound and exhibits a remarkable bioprotective effect against desiccation and heat denaturation of functional protein complexes.

ESEEM measurements were performed at two temperatures on Photosystem I (PS I) complexes depleted of the extrinsic PsaC subunit harboring iron–sulfur 4Fe–4S clusters FA/FB (abbreviated FX-core complexes) embedded in trehalose glassy matrix. The ESEEM modulation frequencies obtained for the FX-core complexes in trehalose matrix were similar at 150 K and 290 K. They correspond to a distance ~ 25 A between the oxidized primary donor P700·+ and reduced phylloquinone acceptor A1·−. This distance corresponds to the preferential charge separation pathway between P700·+ and A1B·− in the B-branch of the PS I redox cofactors, while the distance estimated previously for the intact PS I complexes (containing the extrinsic PsaC subunit) embedded in dry trehalose matrix was ~ 26 A, which is typical for the formation of P700·+A1A·− charge-separated radical pairs in the symmetrical A-branch (Sukhanov et al. in Appl Magn Reson 49:1011, 2018). The redirection of electron transfer preferentially from the A to the B-branch of redox cofactors observed in FX-core complexes embedded in dry trehalose matrix can be rationalized by the alteration of the protein domain rigidity/flexibility around phylloquinone molecules in the A1A and A1B sites, which affects the thermodynamics of electron transfer between P700 and A1 in the symmetrical branches of cofactors. This finding of similar modulation frequencies measured for FX-core complexes in trehalose matrix at 150 K and 290 K is opposite to our previously obtained results for intact PS I, where the value of ESEEM modulation frequency markedly decreased upon heating the sample from cryogenic to room temperature (Sukhanov et al. in Appl Magn Reson 49: 1011, 2018). Our comparison of FX-core complexes with intact PS I complexes suggests that 4Fe-4S clusters accelerate spin–lattice relaxation of the unpaired electron spins on the phylloquinone in the A1-site. The result serves as additional argument in favor of the molecular model of the cryoprotective effect of trehalose matrix on the functioning of PS I by way of rigidity changes of the hydrogen-bonding network of the protein with the matrix that is based on the Le Chatelier–Braun principle. According to this thermodynamic principle, changes in both rigidity and flexibility are compensated to establish new thermodynamic equilibria with restored global balance of rigidity and flexibility that is typical within functioning protein complexes.

Klaus Möbius gives a selection of his biographical experiences which have shaped his academic and personal life.

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., Applied magnetic resonance;Vol. 53. 2022, pages 1239–1263

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.

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.

The one-sided Mobius band topology with its characteristic 180° twist has fascinated and inspired philosophers, artists and scientists since a long time. On the molecular level, only in the last 13 years a few chemistry groups succeeded to artificially create novel compounds with Mobius symmetry by theory-based molecular design and elaborate chemical synthesis. The interest in molecules with Mobius band symmetry was greatly stimulated in 1964 by a theoretical paper by Edgar Heilbronner from the ETH Zurich. 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 into the Mobius topology of their hydrocarbon skeleton. In 2003, the first synthesis of an aromatic Mobius annulene was accomplished by Rainer Herges and co-workers in Kiel. In 2007, Lechoslaw Latos-Grazynski and co-workers in Wroclaw succeeded in synthesizing free-base di-p-benzi (Yoneda et al., Angew Chem Int Ed 53: 13169–13173, 2014) hexaphyrin (1.1.1.1.1.1), compound 1, an expanded porphyrin which can dynamically switch between Huckel and Mobius conjugation upon changes of solvent and temperature. Shortly thereafter, in 2008, Atsuhiro Osuka and his co-workers from Kyoto, Seoul and Hyogo published the synthesis of an expanded porphyrin in which metalation triggered the production of molecular twisting and Mobius aromaticity. In this minireview, among other studies also our recent EPR, ENDOR and DFT studies on open-shell states of 1, i.e., on the ground-state radical cation doublet state (total electron spin S = 1/2) and the first excited triplet state (S = 1) are summarized. The review is largely based on a previous joint publication of the current authors with the Latos-Grazynski group on radical cations of 1 (Mobius et al., Phys Chem Chem Phys 17:6644–6652, 2015). The radical cation study was the first one of an open-shell π-system with Mobius topology. In the doublet state, the hyperfine interactions of the unpaired electron spin with specific magnetic nuclei in the molecule was used as a sensitive probe for the electronic structure of the molecule and its symmetry properties. This work has now been extended to state-of-the-art DFT theory studies on photo-excited triplet states of 1. In the open-shell triplet state, besides hyperfine couplings, a change of the zero-field splitting interaction between the two unpaired electron spins is predicted to be a viable sensor for electronic structure changes upon Mobius-to-Huckel topology switching.

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 structural and dynamical interaction of proteins with their microenvironment in disordered matrices plays a decisive role for their function; EPR spectroscopy is a powerful tool for shading light onto the molecular mechanisms of this protein–matrix interplay. To clarify the molecular mechanisms of disaccharide bioprotection, we studied the structure and dynamics of spin-labeled systems and photosynthetic reaction centers (RCs) in sucrose and trehalose matrices at different hydration levels by means of cw and pulse high-field 95 GHz (W-band) EPR as well as by FTIR. In this minireview, we summarize and discuss EPR and FTIR experiments showing that the anhydrobiotic state of the RC–trehalose system (1) is not the result of matrix-induced changes of the local structure of the charge-separated radical-pair cofactors, $${\text{P}}_{865}^{ \cdot + }$$ and $${\text{Q}}_{\text{A}}^{ \cdot - }$$ , and (2) is not the result of changes of local dynamics and local hydrogen bonding of QA in its binding pocket. Rather, the extreme impairment of RC dynamics caused by incorporation into the dehydrated trehalose matrix, which also protects it against thermal denaturation, originates in the high rigidity, already at room temperature, of the dry trehalose glass matrix coating the RC protein surface. This surface hydrogen-bonding scaffold shifts the correlation time of thermal conformational fluctuations into the non-biological time domain. Another intriguing aspect of disaccharide bioprotection is the superior efficiency of trehalose versus sucrose matrices in stabilizing the anhydrobiotic state of proteins. To clarify the molecular basis of this specificity, glassy trehalose–water and sucrose–water binary systems, incorporating a nitroxide radical as spin probe, have been studied by high-field W-band EPR spectroscopy at different water contents. Analysis of the EPR spectra revealed a different structural and dynamical organization in the sucrose and trehalose matrix, only the trehalose being homogeneous in terms of residual water and nitroxide distribution.

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.

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.

On the basis of experiments at 275 GHz, we reconsider the dependence of the continuous-wave EPR spectra of nitroxide spin-labeled protein sites in sensory- and bacteriorhodopsin on the micro-environment. The high magnetic field provides the resolution necessary to disentangle the effects of hydrogen bonding and polarity. In the gxx region of the 275 GHz EPR spectrum, bands are resolved that derive from spin-label populations carrying no, one or two hydrogen bonds. The gxx value of each population varies hardly from site to site, significantly less than deduced previously from studies at lower microwave frequencies. The fractions of the populations vary strongly, which provides a consistent description of the variation of the average gxx and the average nitrogen-hyperfine interaction Azz from site to site. These variations reflect the difference in the proticity of the micro-environment, and differences in polarity contribute marginally. Concomitant W-band ELDOR- detected NMR experiments on the corresponding nitroxide in perdeuterated water resolve population-specific nitrogen-hyperfine bands, which underlies the interpretation for the proteins.

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.

Photosystem I (PS I) mutants from the cyanobacterium Synechocystis sp. PCC 6803 bearing point mutations to the axial ligands of A0A (M688NPsaA) and A0B (M668NPsaB) were studied by high-field W-band electron paramagnetic resonance (EPR) spectroscopy. It was found that the EPR observables of PS I from the M668NPsaB mutant were virtual identical to that of the wild type (WT), and are clearly distinct from the M688NPsaA mutant. In particular, the P700·+ decay kinetics in the M688NPsaA mutant is significantly slower than in the WT or the M668NPsaB mutant. The analysis of the out-of-phase electron–electron dipolar electron spin echo envelope modulation shows that in the M668NPsaB mutant, the estimated distance of 26.0 ± 0.3 A agrees well with the 25.8 A distance for the P700·+A1A·− radical pair measured in the X-ray crystal structure. In the M688NPsaA mutant, two populations are found with estimated distances of 26.0 ± 0.3 and 25.0 ± 0.3 A in a ratio of 0.7–0.3, which agree well with the 25.8 A distance for the P700·+A1A·− radical pair and the 24.6 A distance for the P700·+A1B·− radical pair measured in the X-ray crystal structure. The data confirm that under the experimental conditions employed in this work, which involve dark-adapted samples without the pre-reduction of the iron–sulfur clusters, electron transport in cyanobacterial PS I is asymmetrical at 100 K, with the majority of electron transfer taking place through the A-branch of cofactors.

High-field W-band electron paramagnetic resonance (EPR) spectroscopy was utilized to study the temperature dependence of the magnetic interaction parameters (g-, hyperfine-, quadrupole tensors) of two types of doublet-state nitroxide spin probes in glass-forming ortho-terphenyl solution: a five-membered ring system of pyrroline type (model for the commonly used methane thiosulfonate spin label) and a six-membered ring system of piperidine type (model for the commonly used TOAC spin label). The analysis of the g- and hyperfine tensors in terms of their isotropic and anisotropic parts reveals at least two mechanisms of motion that are responsible for the temperature dependence of the interaction parameters. The first mechanism is attributed to the overall small-angle motion of the nitroxide molecule in the glassy matrix; it leads to an averaging of the anisotropies of the EPR parameters. The second mechanism originates in an intramolecular out-of-plane motion of oxygen in the nitroxide group. This type of motion is evidenced by comparing the experimental findings for the spin-interaction parameters with the results of density functional theory calculations. The harmonic oxygen out-of-plane vibrations result in a variation of both the isotropic and anisotropic parts of the g- and hyperfine tensors. In contrast, the quadrupole tensor is not influenced by this vibration mechanism in the temperature range under study (90–240 K). Consequences of the applicability of such typical nitroxide radicals for probing details of their protein environment and for studying librational dynamics in frozen solutions are discussed.

In a recent paper (Bardelang et al. in J Org Chem 71:7657–7667, 2006), the deep inclusion of the TIPNO (1-phenyl-2-methylpropyl-1,1-dimethylethyl-2-nitroxide) free radical attached to a permethylated β-cyclodextrin (TRIMEB) into its cavity was questioned. In the present paper it is shown that the results of X-band and W-band electron paramagnetic resonance (EPR) and 1H nuclear magnetic resonance (NMR) studies of the TIPNO–TRIMEB in the presence of competitors of complexation (1-adamantanol, methyl orange and 1-adamantylamine) are consistent with a situation where the nitroxide is capping the small cavity entrance. The inclusion of the incoming guest was proven by 1H NMR, whereas no changes in EPR spectra were noticed whatever the competitors’ concentrations. These observations are rationalized in terms of an equilibrium between a nitroxide-capped cyclodextrin (weak complex) and a non-self included form, both species involving competitors’ inclusion without significant EPR spectral changes. These results not only confirm our preliminary findings, but also point out to the peculiar role of the cyclodextrin methoxy crown of the primary rim stabilizing the appended guest by means of weak interactions.

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.

The combination of high-field electron paramagnetic resonance (EPR) with site-directed spin labeling (SDSL) techniques employing nitroxide radicals has turned out to be particularly powerful in revealing subtle changes of the polarity and proticity profiles in proteins enbedded in membranes. This information can be obtained by orientation-selective high-field EPR resolving principal components of the nitroxide Zeeman (g) and hyperfine ( A) tensors of the spin labels attached to specific molecular sites. In contrast to the g- and A-tensors, the (14)N ( I = 1) quadrupole interaction tensor of the nitroxide spin label has not been exploited in EPR for probing effects of the microenvironment of functional protein sites. In this work it is shown that the W-band (95 GHz) high-field electron spin echo envelope modulation (ESEEM) method is well suited for determining with high accuracy the (14)N quadrupole tensor principal components of a nitroxide spin label in disordered frozen solution. By W-band ESEEM the quadrupole components of a five-ring pyrroline-type nitroxide radical in glassy ortho-terphenyl and glycerol solutions have been determined. This radical is the headgroup of the MTS spin label widely used in SDSL protein studies. By DFT calulations and W-band ESEEM experiments it is demonstrated that the Q(yy) value is especially sensitive to the proticity and polarity of the nitroxide environment in H-bonding and nonbonding situations. The quadrupole tensor is shown to be rather insensitive to structural variations of the nitroxide label itself. When using Q(yy) as a testing probe of the environment, its ruggedness toward temperature changes represents an important advantage over the g xx and A(zz) parameters which are usually employed for probing matrix effects on the spin labeled molecular site. Thus, beyond measurenments of g xx and A(zz) of spin labeled protein sites in disordered solids, W-band high-field ESEEM studies of (14)N quadrupole interactions open a new avenue to reliably probe subtle environmental effects on the electronic structure. This is a significant step forward on the way to differentiate between effects from matrix polarity and hydrogen-bond formation.

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.

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 1 B • − → F X ${\rm{A}}_{{\rm{1A}}}^{ \bullet - }/{\rm{A}}_{1{\rm{B}}}^{ \bullet - } \to {{\rm{F}}_{\rm{X}}}$ by time-resolved optical spectroscopy at 480 nm. The kinetics of the primary ET reactions to form the primary P 700 • + A 0 • − ${\rm{P}}_{700}^{ \bullet + }{\rm{A}}_0^{ \bullet - }$ and the secondary P 700 • + A 1 • − ${\rm{P}}_{700}^{ \bullet + }{\rm{A}}_1^{ \bullet - }$ ion radical pairs were not affected by dehydration in the trehalose matrix, while the yield of the P 700 • + A 1 • − ${\rm{P}}_{700}^{ \bullet + }{\rm{A}}_1^{ \bullet - }$ was decreased by ~20%. Forward ET from the phylloquinone molecules in the A 1 A • − ${\rm{A}}_{1{\rm{A}}}^{ \bullet - }$ and A 1 B • − ${\rm{A}}_{1{\rm{B}}}^{ \bullet - }$ 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 1 B • − ${\rm{A}}_{1{\rm{B}}}^{ \bullet - }$ and P 700 • + . ${\rm{P}}_{700}^{ \bullet + }.$ 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.

An improved TM110 resonator for continuous-wave and time-resolved electron-nuclear double resonance (ENDOR) studies at X-band frequencies is described that has been designed with small samples and/or light excitation in mind. The filling factor is increased by reducing the resonator length to only 16 mm. The radio-frequency field is generated by either a conventional solenoid for stable samples or by a pair of coils for samples which require photoexcitation. This arrangement leaves the central volume of the resonator free for optimal sample illumination, which is achieved through a slit in the wall of the resonator. Microwave coupling is achieved by the incorporation of an iris in the top of the resonator, and magnetic field modulation is applied by external coils. The resonator’s high sensitivity is illustrated by a study of the temperature dependence of the continuous-wave ENDOR signal of the stable neutral flavin radical in DNA photolyase and an investigation of the photoexcited triplet state of free-base tetraphenylbacteriochlorin by time-resolved ENDOR.

In this work, by using the respective advantages of W- and X-band electron paramagnetic resonance (EPR) spectroscopy techniques to investigate electron transport processes, we have studied the light-induced redox transients of the primary electron donor P700 and the secondary acceptor A1 in photosystem (PS) I complexes of intact cyanobacterial cellsSynechocystis sp. PCC 6803. We found that the kinetic behavior of the cation radical P700·+ generated by illumination with continuous light, and the EPR intensity of the radical pair P700·+A1·− generated upon laser pulse illumination strongly depend on the illumination prehistory (either the sample was frozen in the dark or during illumination). Both these processes were sensitive to the presence of electron transport inhibitors which block electron flow between the two photosystems. In line with our X-band EPR data on the kinetics of light-induced redox transients of P700, our high-field W-band EPR study of the radical-pair state P700·+A1·− shows that photosynthetic electron flow through the PS I reaction center is controlled both on the donor and on the acceptor side of PS I.

We report on recent 95 and 360 GHz high-field electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR) and pulsed electron-electron double resonance (PELDOR) studies of wild-type and mutant reaction centers (RCs) from the photosynthetic bacteriumRhodobacter sphaeroides. Taking advantage of the excellent spectral and temporal resolution of EPR at 95 and 360 GHz, the electron-transfer (ET) cofactors radical ions and spin-correlated radical pairs were characterized by theirg- and hyperfine-tensor components, their anisotropicT2 relaxation as well as by the dipolar interaction between P865•+QA•− radical pairs. The goal of these studies is to better understand the dominant factors determining the specificity and directionality of transmembrane ET processes in photosynthetic RC proteins. In particular, our multifrequency experiments elucidate the subtle cofactor-protein interactions, which are essential for fine-tuning the ET characteristics, e.g., the unidirectionality of the light-induced ET pathways along the A branch of the RC protein. By our high-field techniques, frozen-solution RCs of novel site-specific single and double mutants ofR. sphaeroides were studied to modulate the ET characteristics, e.g., even to the extent that dominant B branch ET prevails. The presented multifrequency EPR work culminates in first 360 GHz ENDOR results from organic nitroxide radicals as well as in first 95 GHz high-field PELDOR results from orientationally selected spin-polarized radical pairs P865•+QA•−, which allow to determine the full geometrical structure of the pairs even in frozen-solution RCs.

Matrix effects on long-range electron transfer were studied in cyanobacterial Photosystem I (PS I) complexes, embedded into trehalose glasses at different hydration levels. W-band EPR studies demonstrated, via nitroxide spin probes, structural homogeneity of the dry PS I-trehalose matrix and no alteration of cofactors' distance and relative orientation under temperature and matrix variation. In dry trehalose glasses at room temperature (RT), PS I was stable for months. Flash-induced charge recombination kinetics were examined by high-field time-resolved EPR and optical spectroscopies. The kinetics in hydrated PS I-trehalose glasses mostly reflected the reduction of the photooxidized primary donor P700•+ by the reduced terminal iron-sulfur clusters. Upon dehydration, the P700•+ decay accelerated and became more distributed. Continuous distributions of lifetimes τ were extracted from the kinetics by two numerical approaches: a maximum entropy method (MemExp program) and a constrained regularization method (CONTIN program). Both analyses revealed that upon dehydration the contribution of the two slowest components (lifetimes τ ~ 300 ms and ~ 60 ms), attributed to P700•+[FA/FB] − recombination, decreased in parallel with the increase of the fastest component (τ ~ 150 μs), and of additional distributed phases with intermediate lifetimes. Dehydration at RT mimicked the effects of freezing water-glycerol PS I systems, suggesting an impairment of PS I protein dynamics in the dry trehalose glass. Similar effects were observed previously in bacterial reaction centers. The work presented for PS I provides new insights into the crucial issue of protein-matrix interactions for protein functionality as controlled by hydrogen-bond networks of the hydration shell.

A computational model to simulate electron spin polarization in the three-spin-1/2 system composed of the molecular excited triplet state of (tetraphenylporphinato)zinc(II) (ZnTPP) and the doublet ground state of the 3-(N-nitronyl-notroxide) pyridine (3-NOPy) stable radical is proposed. The model is based on numerical solutions of the stochastic Liouville equation for the diffusively rotating system where the magnetic dipolar, isotropic Heisenberg exchange, and anisotropic Zeeman electron spin interactions are taken into account in a full measure, whereas the intersystem crossing processes between the singlet and triplet states of ZnTPP are considered in terms of kinetic equations for the relevant spin density matrices. Additional longitudinal and transversal paramagnetic relaxation caused by relative rotation motions of the ZnTPP and 3-NOPy moieties is taken into consideration in the form of the generalized relaxation operator.

The g-tensor of the neutral radical form of the flavin adenine dinucleotide cofactor FADH• of (6–4) photolyase from Xenopus laevis has been determined by very high-magnetic-field/high-microwave-frequency electron-paramagnetic resonance (EPR) performed at 360 GHz/12.8 T. Due to the high spectral resolution the anisotropy of the g-tensor could be fully resolved in the frozen-solution continuous-wave EPR spectrum. By least square fittings of spectral simulations to experimental data, the principal values of the g-tensor have been established: gX = 2.00433(5), gY = 2.00368(5), gZ = 2.00218(7). A comparison of very high-field EPR data and proton and deuteron electron-nuclear double resonance measurements yielded precise information concerning the orientation of the g-tensor with respect to the molecular frame. This data allowed a comparison to be made between the principal values of the g-tensors of the FADH• cofactors of photolyases involved in the repair of two different DNA lesions: the cyclobutane pyrim...

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.

A vacuum-tube device for the generation of pulsed microwave radiation in the submillimeter range (up to 380 GHz) is presented, designed for use as a source in a 360 GHz high-field/high-frequency electron paramagnetic resonance (EPR) spectrometer—the pulsed Orotron. Analogous to the known continuous wave (cw) version, in the pulsed Orotron microwave radiation is generated by the interaction of a nonrelativistic electron beam with a diffraction grating (stimulated Smith–Purcell radiation) in feedback with an open Fabry–Perot resonator construction. The presented design extends the cw Orotron by a gate electrode and a high-voltage pulsing unit to control the electron beam current. The generated pulses at 360 GHz have pulse lengths from 100 ns–10 μs and a pulse power of (22±5) mW. The output in a broader frequency band between 320 and 380 GHz ranges from 20 up to 60 mW. Within a 10 μs time slot, incoherent pulse trains of arbitrary duration can be generated. The pulsed Orotron has been incorporated in the quasioptical microwave bridge of a heterodyne induction mode EPR spectrometer. The first free induction decay measurements at a microwave frequency of 360 GHz and a magnetic field of 12.8 T on a polycrystalline perylenyl–ion sample are presented and future applications and extensions of Orotron-EPR spectroscopy are discussed.

The pore-forming bacterial toxins of the colicin family undergo massive protein refolding, while attacking a target cell, to convert from the water-soluble conformational state to the membrane-associated state with subsequent insertion of helical hairpins into the cytoplasmic membrane. To explore the validity of proposed models for the mechanism by which the soluble channel-forming domain of colicin A turns inside out upon membrane association, five site-specific cysteine mutants of colicin A, each singly spin labeled with nitroxide side chains, were studied by 9.5 GHz (X-band) and 95 GHz (W-band) high-field EPR. By elucidating the mobility of the nitroxide side chains on one of the two hydrophobic helices and their accessibility to paramagnetic relaxer molecules in the membrane, as well as by measuring the gxx and Azz nitroxide tensor components, detailed information about conformational changes upon membrane association could be revealed. This information on the channel-forming domain of colicin A goes ...

The frozen solution electron paramagnetic resonance spectrum of the primary donor cation P + in reaction centers of site-directed mutants of Rhodobacter ( Rb. ) sphaeroides has been obtained at a microwave frequency ν =360 GHz and a magnetic field B 0 =12.8 T. Due to the high Zeeman resolution of the powder pattern, all three principal components of the rhombic g -tensors at T =160 K could be determined with high accuracy. We compare spectra of the site-directed mutants, in which the axial ligand histidine M202 of the primary donor is replaced by glutamic acid (HE(M202)) or leucine (HL(M202)), with those of the strain R26, whose primary donor is similar to that of the wild type and only lacks the carotenoid. For HE(M202), this is the first determination of its g -tensor with the principal components g xx =2.00335(3), g yy =2.00236(2) and g zz =2.00191(2). While in R26 the primary donor is a bacteriochlorophyll a dimer, the HL(M202) and HE(M202) mutants have previously been shown to be bacteriochlorophyll:bacteriopheophytin heterodimers. Their g -tensor anisotropy Δ g = g xx − g zz shows significant variations in opposite directions when compared with R26, with an increased anisotropy for HE(M202) and a decreased one for HL(M202). Calculations employing Density Functional Theory suggest that the observed shifts originate in different torsional angles of the acetyl group attached to the spin-carrying bacteriochlorophyll half L of the dimer.

EPR spectroscopy at 95 GHz was used to characterize the dynamics at the Mn2+ binding site in single crystals of the saccharide-binding protein concanavalin A. The zero-field splitting (ZFS) tensor of the Mn2+ 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 Mn2+ site, at low temperatures two types of Mn2+ 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 Mn2+ 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 s...

Hotly debated issues in the area of biological electron transfer (ET) involve questions of how long-range ET processes proceed along pathways that incorporate non-covalently linked cofactors interacting with their protein microenvironment. To contribute answers to such questions, time-resolved EPR (TREPR) experiments at various microwave frequency/magnetic field settings have been performed. In this overview, representative examples of this work from our laboratory are given which comprise transient intermediates of light-induced ET in (i) bacterial photosynthetic reaction centers and (ii) biomimetic model complexes. In these complexes the donor and acceptor components are tethered together either covalently via cyclohexenyl bridges or non-covalently via Watson-Crick base-pairing. The studies are aiming at broadening our knowledge of structure-dynamics-function relationships associated with ET processes. It is demonstrated that high-field TREPR opens new perspectives in elucidating complex photochemical E...