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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.

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.

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.

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.

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...

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.

Semiempirical molecular orbital methods (PM3, INDO, ZINDO/S) have been used to calculate the effects of local electric fields and of hydrogen bonding on the g and hyperfine tensors of a nitroxide spin label model system. The results yield a linear correlation between the two principal tensor components g xx and A N zz at label sites of varying polarity. Hydrogen bonding with a single water molecule produces a constant shift of Δg xx ≅ −4 × 10−4. These theoretical results are used to interpret recent high field (3.4 T, 95 GHz) electron paramagnetic resonance investigations on site-directed spin labelled bacteriorhodopsin. This protein reveals a close correlation between proticity and polarity at the various label sites. The slope of the g xx versus A N zz dependence is affected strongly by polarity induced structural strains of the spin label.

The radical-pair state of the primary electron donor and the secondary electron acceptor (P(700)(+z.rad;)A(1)(-z.rad;)) of the photosynthetic reaction center (RC) photosystem I (PS I) of Synechocystis PCC 6803 was studied by time-resolved electron paramagnetic resonance (TREPR) at high field/high frequency (3.4 T/95 GHz) using orientation selection in multilayers. The goal of the present article is to work out the basis for future studies, in which the improved resolution of such multilayers may be used to detect mutation-induced structural changes of PS I in membrane preparations. This approach is particularly interesting for systems that cannot be prepared as single crystals. However, in order to use such multilayers for structural investigations of protein complexes, it is necessary to know their orientation distribution. PS I was chosen as a test example because the wild type was recently crystallized and its X-ray structure determined to 2.5 A resolution [Nature 411 (2001) 909]. On the basis of our experimental results we determined the orientation distribution. Furthermore, a simulation model for the general case in which the orientation distribution is not axially symmetric about the C(2) symmetry axis of the RC is developed and discussed. Spectra simulations show that changes in the TREPR spectra of PS I are much more significant for these oriented multilayers than for disordered samples. In this way the use of oriented multilayers, in conjunction with multifrequency TREPR measurements on oriented as well as on disordered samples, is a promising approach for studies of structural changes of PS I systems that are induced by point mutations.

The flavin cofactor of Escherichia coli DNA photolyase in its neutral radical form, FADH•, was investigated by high-frequency/high-field continuous-wave electron paramagnetic resonance at 360 GHz. The data presented are the first flavin radical spectra where the full rhombic symmetry of the g-tensor is resolved. A fit of the spectrum yields accurate principal values of g, which show only a small anisotropy: gX = 2.004 31(5), gY = 2.003 60(5) and gZ = 2.002 17(7). The hyperfine splitting observed in the gY region could be assigned to a hyperfine tensor component of the H(5) proton in the 7,8-dimethyl isoalloxazine moiety of FADH•. From a comparison of this effective hyperfine coupling with the principal value obtained from pulsed (Davies) electron−nuclear double resonance, the orientation of the g-tensor principal axes with respect to the H(5) hyperfine principal axes could be derived. Remaining ambiguities in the sign of the angle between the principal axes of g and the molecular axes are discussed by ta...

Continuous-wave high-field electron paramagnetic resonance (95 GHz, 3.4 T) is performed on a spin label side chain located at residue position 171 in the proton entrance channel of bacterior-hodopsin The conformational differences of three bacteriorhodopsin mutants, the single mutant F171C, the double mutant D96G/F171C, and the triple mutant D96G/F171C/F219L, are reflected in different gxx and Azz tensor component shifts of the nitroxide side chain. The most polar microenvironment is found in the single mutant, whereas the open proton entrance channel reported for the triple mutant allows a reorientation of the nitroxide group towards a microenvironment of lower polarity and/or reduced hydrogen bonding. The experimental data of the double mutant are explained by a light-independent equilibrium of two nitroxide orientations with different polarities of the local microenvironment. Upon illumination the spectrum of the single mutant revealsg xx andA zz tensor component shifts which resemble those determined for the triple mutant in the dark. This result provides strong evidence for a light-induced opening of the proton entrance channel of the single mutant similar to that found in the unilluminated triple mutant, in agreement with electron diffraction data.

The photoexcited quartet (Q1) and doublet (D1) states of the complex of tetra-phenyl-porphine zinc (II) and a nitroxide radical have been studied in toluene solution by X-(9.4 GHz) and W-band (94 GHz) time-resolved electron paramagnetic resonance spectroscopy. The spin-polarized signals of the Q1 and D1 states are observed and assigned by analysis of theg-values. Line broadening and spin polarization mechanisms in this system are discussed.

Density functional theory is used to calculate the electronic structure of the neutral flavin radical, FADH(*), formed in the light-induced electron-transfer reaction of DNA repair in cis,syn-cyclobutane pyrimidine dimer photolyases. Using the hybrid B3LYP functional together with the double-zeta basis set EPR-II, (1)H, (13)C, (15)N, and (17)O isotropic and anisotropic hyperfine couplings are calculated and explained by reference to the electron densities of the highest occupied molecular orbital and of the unpaired spin distribution on the radical. Comparison of calculated and experimental hyperfine couplings obtained from EPR and ENDOR/TRIPLE resonance leads to a refined structure for the FAD cofactor in Escherichia coli DNA photolyase. Hydrogen bonding at N3H, O4, and N5H results in significant changes in the unpaired spin density distribution and hyperfine coupling constants. The calculated electronic structure of FADH(*) provides evidence for a superexchange-mediated electron transfer between the cyclobutane pyrimidine dimer lesion and the 7,8-dimethyl isoalloxazine moiety of the flavin cofactor via the adenine moiety.

We investigate the effect of stochastic modulation of the exchange interaction Jex on singlet (S)-triplet (T) transitions in radical pairs. These transitions limit the lifetime of the photo-generated radical pairs in covalently linked porphyrin-quinone systems that have been developed for biomimetic modeling of photosynthetic electron transfer processes. In order to explain transient electron paramagnetic resonance (EPR) results in different magnetic fields, i.e., with X-band ((0.34 T)/ (9.5 GHz)) and W-band ((3.4 T)/(95 GHz)) time-resolved EPR, we have to assume that Jex is modulated over a range of 20000 G, which is wide enough that S is temporarily almost degenerate with T0 as well as with T−1. This large modulation of Jex is caused by restricted rotational diffusion of the quinone subunit with respect to the porphyrin subunit. However, because of the small interradical distance of about 1.0–1.4 nm, the radical pair is continuously kept in the strong coupling limit and, therefore, we observe only EPR transition between the triplet sublevels. We find an approximation to solve the stochastic Liouville equation valid for rotational diffusion on an intermediate time scale, i.e., the diffusion rate DR is smaller than the singlet electron recombination rate KS ∼ 109 s−1, but larger than the ST transition rates κ0, κ−1 < 106 s−1.

Primary photosynthesis is the biological electron transfer process by which green plants and certain bacteria convert the energy of sunlight into electrochemical energy. Light-induced charge separation is achieved by (bacterio) chlorophyll donor and quinone acceptor cofactors in the transmembrane reaction center protein complexes. Thereby transient radical ions are formed creating weakly coupled radical pairs. Time-resolved high-field EPR spectroscopy is ideally suited to study such short-lived species in their working states. It provides detailed information on the structure and dynamics of the cofactors in their binding sites and on hydrogen bond interaction with the protein. Thereby, our understanding of primary photosynthesis on the molecular level is improved.

The photoexcited triplet state of free-base porphycene randomly oriented in a glassy toluene matrix has been investigated by time-resolved electron paramagnetic resonance and electron spin echo spectroscopy over the temperature range 4–100 K. Spectral broadening and anisotropic relaxation are both observed as the temperature is raised, demonstrating the complementary use of these techniques. It is concluded that two triplet states are present in the EPR spectra: only one at 4 K, and the other becoming populated at higher temperatures. This behaviour is explained in terms of intramolecular N–H tautomerism in the porphycene macrocycle. The N–H tautomerism is estimated to have a maximum rate of 7×108 s-1 at 100 K.

Using W-band time-resolved EPR (TREPR) (95 GHz), the lowest excited doublet (D1) state was observed in solution for the first time together with the excited quartet (Q1) and doublet ground (D0) states in a complex of MgTPP (TPP; tetraphenylporphyrin) and an axial-ligating p-pyridylnitronylnitroxide radical (nit-p-py). The g value obtained for D1 was in good agreement with the calculated value. The spin polarization of D1 varied with time from an emission to an absorption as well as that of Q1 and D0. The result shows that the spin polarizations are produced by a radical−triplet pair mechanism with a ferromagnetic coupling (J > 0). The sign of the exchange coupling is consistent with that obtained in the X-band TREPR experiment (Fujisawa, J.; Ishii, K.; Ohba, Y.; Yamauchi, S.; Fuhs, M.; Mobius K. J. Phys. Chem. A 1997, 101, 5869).

Time-resolved electron paramagnetic resonance (EPR) investigations, performed at microwave frequencies of 9.5 GHz (X band) and 95 GHz (W band), on porphyrin quinone quinone and porphyrin porphyrin quinone triads are presented. Time-resolved EPR at different microwave frequencies makes it possible to elucidate the electron transfer (ET) reactions after pulsed laser excitation and to determine their intermediates, the charge-separated radical pair (RP) states. After photoexcitation in the nematic and soft glass phase of a liquid crystal (LC), spin-polarized EPR spectra are observed for the RPs and for the triplet states of the porphyrins created by spin-selective intersystem crossing (ISC) from the excited porphyrin singlet state. The EPR polarization patterns of the RPs are discussed in terms of the favoured decay channels of the photoexcited singlet state of the porphyrin donor. The decay pathway can be singlet ET to the quinone(s) followed by singlet/triplet mixing to yield RPs with triplet character, or...

The photoexcited triplet state of free-base porphycene randomly oriented in a glassy toluene matrix has been investigated by time-resolved EPR over the temperature range 4-200 K. The spectra show a remarkable temperature dependence. It is concluded that two triplet states are present in the EPR spectra: only one is present at 4 K, and the other is populated at higher temperatures. The energy difference between the two states is estimated to be 0.4 kJ mol-1. The behaviour is explained in terms of fast intramolecular NH tautomerism present in the porphycene macrocycle. As the temperature is raised the exchange between the two states increases and one effective state is observed. In addition, at even higher temperatures where the solvent is close to the liquid-glass transition, the triplet spectrum is affected by anisotropic relaxation caused by rotation of the porphycene molecule in the molecular plane.

Davies-type pulsed H electron nuclear double resonance (ENDOR) measurements were performed at a magnetic field of 3.4 T and a microwave (MW) frequency of 95 GHz (W-band). By taking advantage of the increased electron Zeeman interaction at high field, the small g-anisotropy of the semiquinone anion radicals could be resolved in frozen solutions. Hence, the W-band ENDOR spectra could be taken at the well-separated canonical peaks of the powder electron paramagnetic resonance (EPR) spectra, thereby becoming highly orientation-selective with respect to the relative orientation of the radicals to the external magnetic field. The measurements were performed on various randomly oriented semiquinone anion radicals in frozen alcoholic solution and on the primary ubiquinone anion radical, Q, frozen photosynthetic bacterial reaction centers (RCs) of Rhodobacter sphaeroides in which the Fe was replaced by Zn (ZnRC). A simulation program was used to obtain magnitudes and orientations of the hyperfine tensors. The W-band ENDOR spectra of the immobilized radicals show not only hyperfine couplings (HFC) of local protons of the semiquinones, but also those of protons from the environment. These are, for example, involved in hydrogen bonds between the amino acid surrounding and the quinone carbonyl groups. For Q in ZnRCs, a particularly large H-bond HFC was obtained from which direction and distance of the H-bond could be estimated. These data were compared with those measured for the respective ubisemiquinone radical, UQ-10, in protonated and deuterated 2-propanol, where two H-bonds of comparable strength but different directions could be detected. Comparison of H ENDOR spectra of the 2,3,5,6-tetra-methyl-1,4-benzoquinone anion radical in frozen solution, performed both at W-band and at X-band frequency, demonstrated the limitations of achieving orientation selected ENDOR spectra from X-band experiments on systems with small g-anisotropy.

The “Contracted Porphyrins” corrole and the recently synthesized isocorrole (as octaethyl derivatives) represent novel porphyrinoid macrocycles of biochemical interest. The radical cations of the free-bases of both corrole and its isomer are studied by EPR, ENDOR, and TRIPLE resonance in liquid solution to measure isotropic hyperfine couplings including signs. They yield detailed information of the electronic structure of the radical cations. The experimental findings are compared with results of all-valence-electrons self-consistent field molecular orbital calculations (RHF-INDO/SP). The comparison shows that the free-bases of corrole and isocorrole undergo protonation when oxidized to cation radicals in CH2Cl2 solution. Excellent agreement of the measured and calculated spin density distributions is achieved for the protonated dication radicals, which exhibit the C2 symmetry observed for the hyperfine structure. The analysis of the hyperfine data discriminates against NH tautomerism as the sole source for an effective C2 symmetry of the macrocycle.

ENDOR spectra of triplet state molecules have a chacteristic line from degenerate NMR transitions within the zero level (ZL) electron spin manifoldM S=0. The ZL line, observed at the free nuclear Larmor frequency, dominates spectra when the number of nuclei is large. This line was found to be substantially reduced in intensity at low temperature. The strong variation of the ZL line intensity is analyzed within the frame of an electric-circuit analogy modeling. The result is as follows: At low temperature the electron and nuclear spin-lattice relaxation rates become small, and the nuclear-nuclear spin flip-flop transitions between degenerate substates,M I=const within the ZL manifold, become relatively strong to compete for population redistribution. This reduces the population differences between nuclear sublevels. Additional NMR irradiation can thus do very little to reduce these differences even more, and the ENDOR effect becomes suppressed. A certain enhancement of the relaxationally suppressed ZL line occurring at increased EPR saturation is explained by the coherent action of the microwave field on the selected substate within the ZL manifold that shifts its energy out of the other degenerate states thereby closing the flip-flop relaxation channel.

The specific ENDOR line at the free Larmor frequency νp in the low temperature spectra of triplet state molecules is caused by degenerate NMR transitions within theMs=0 zero-level (ZL) electron spin manifold. This ZL line was found to be orientationally dependent for the diradical complex Zn(3,6-di-tert-butyl-o-semiquinone)2Zn(DBSQ)2: the ZL line dominates the ENDOR spectrum if it is detected at the perpendicular canonical components of the EPR spectrum, and vanishes if the complex is oriented with its ZFSz-axis parallel to the direction of the magnetic field, i.e., if detected at the parallel canonical EPR components. This effect is shown to result from the interaction between nuclear spin substates of the S and T0 manifolds, their levels being close to each other for the Zn(DBSQ)2 complex. Such an interaction mixes the states and shifts energy levels. Consequently, it cancels the degeneracy of the nuclear substates within the ZL manifold and reduces the rate of nuclear flip-flop relaxation. This specific relaxation mechanism has been shown to substantially affect the amplitude of the ZL line (Doubinskii A.A., Lebedev Ya.S., Mobius K.: Appl. Magn. Reson.13, 439 (1997)). The nuclear flip-flop relaxation effect is expected to be orientationally dependent since the S-To separation depends upon the orientation of the diradical with respect to the external magnetic field.

The electronic structure of the primary electron donor (D) of the photosynthetic bacteriumRhodobacter sphaeroides is investigated by ENDOR in the photoinduced triplet state of D. Hyperfine (hf) splittings of the triplet state measured on frozen solutions are given and compared to the results obtained earlier (Lendzian F., van Willigen H., Sastry S., Mobius K., Scheer H., Feick R.: Chem. Phys. Lett.118, 145 (1985). The hf splittings found are consistent with a model of an asymmetric spin density distribution over the two bacteriochlorophyll molecules which constitute D, suggesting a mirror image symmetry of HOMO and LUMO coefficients. The could be relevant for electron transfer, in particular unidirectionality in the reaction center (RC). The first triplet state ENDOR experiments on single crystals of RC’s are also reported.

The bacteriochlorophyll (BGhl)a cation radical is studied by cw-EPR and multinuclear ENDOR/TRIPLE resonance techniques in liquid solution yielding the hyperfine coupling constants (hfcs) for 12 protons, all 4 nitrogens and the central magnesium nucleus. The H hfcs are assigned to specific molecular positions by various experimental techniques including isotope substitution, H/D exchange experiments and comparison with bacteriochlorophyll derivatives. The determined temperature dependences of the H hfcs are measured and related to dynamical properties of the molecule in solution. The experimental hfcs compare well with those obtained from semiempirical molecular orbital calculations of the RHF-INDO/SP type. The importance of these results for BChl speciesin vivo, i.e., in the protein matrix, is discussed.