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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.

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.

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.

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.

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.

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...

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.

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.

The spin-polarized EPR spectra at 95 GHz (W-band), 24 GHz (K-band), and 9 GHz (X-band) of the radical pair in highly purified photosystem I particles are presented. The spectra are analyzed to obtain both the magnetic parameters of the radical pair as well as the relative orientation of the two species. From the analysis, the g-tensor of is found to be gxx = 2.0062, gyy = 2.0051, and gzz = 2.0022, and it is shown that A1 is oriented such that the carbonyl bonds are parallel to the vector joining the centers of and . The anisotropy of the g-tensor is considerably larger than that obtained for chemically reduced phylloquinone in frozen 2-propanol solution. Possible reasons for this difference and their implications for the A1 binding site are discussed. The relative orientation of and is compared with earlier estimates obtained using less accurate g-values for . A comparison with the spectra of in bacterial reaction centers (bRCs) of Rhodobacter sphaeroides R-26 in which the nonheme iron has been replaced b...

Some organisms can survive complete dehydration and high temperatures by adopting an anhydrobiotic state in which the intracellular medium contains large amounts of disaccharides, particularly trehalose and sucrose. Trehalose is most effective also in protecting isolated in vitro biostructures. In an attempt to clarify the molecular mechanisms of disaccharide bioprotection, we compared the structure and dynamics of sucrose and trehalose matrices at different hydration levels by means of high-field W-band EPR and FTIR spectroscopy. The hydration state of the samples was characterized by FTIR spectroscopy and the structural organization was probed by EPR using a nitroxide radical dissolved in the respective matrices. Analysis of the EPR spectra showed that the structure and dynamics of the dehydrated matrices as well as their evolution upon re-hydration differ substantially between trehalose and sucrose. The dehydrated trehalose matrix is homogeneous in terms of distribution of the residual water and spin-probe molecules. In contrast, dehydrated sucrose forms a heterogeneous matrix. It is comprised of sucrose polycrystalline clusters and several bulk water domains. The amorphous form was found only in 30% (volume) of the sucrose matrix. Re-hydration leads to a structural homogenization of the sucrose matrix, whilst in the trehalose matrix several domains develop differing in the local water/radical content and radical mobility. The molecular model of the matrices provides an explanation for the different protein–matrix dynamical coupling observed in dried ternary sucrose and trehalose matrices, and accounts for the superior efficacy of trehalose as a bioprotectant. Furthermore, for bacterial photosynthetic reaction centers it is shown that at low water content the protein–matrix coupling is modulated by the sugar/protein molar ratio in sucrose matrices only. This effect is suggested to be related to the preference for sucrose, rather than trehalose, as a bioprotective disaccharide in some anhydrobiotic organisms.

The distance and relative orientation of functional groups within protein domains and their changes during chemical reactions determine the efficiency of biological processes. In this work on electron transfer proteins, we report the results of orientation resolving dipolar high-field EPR spectroscopy on the charge-separated state P700•+ A1•– (P700, primary electron donor; A1, phylloquinone electron acceptor) in Photosystem I (PS I). Pulsed high-field EPR spectroscopy at W-band (95 GHz, 3.4 T) with extensions to PELDOR (pulsed electron–electron double resonance) and RIDME (relaxation-induced dipolar modulation enhancement) was utilized to obtain the parameters describing the three-dimensional structure of the laser-flash-induced transient radical pair P700•+ A1•– in a frozen solution of deuterated PS I from the cyanobacterium Synechocystis sp. PCC 6803, which is performing oxygenic photosynthesis. The measured distances and relative orientations of the weakly coupled radical ions in the radical pair P700•+ A1•– are compared with previously reported geometries and with those of the precursor cofactors P700 and A1 known from X-ray crystallography. Cyclic electron transfer was found to proceed exclusively via the A-branch of the cofactor chain of PS I at cryogenic temperature. The position and orientation of the reduced phylloquinone coincide with those of the precursor, revealing that no substantial orientational changes of the phylloquinone molecule upon charge separation occur. Several distinct orientations of the P700•+ g-tensor axes with respect to the molecular frame of the primary donor were found experimentally, which we explain by several conformational substates of the P700•+ radical structure having slightly different electron spin density distributions.

Cation radicals of the primary electron donor (D + ) in bacterial photosynthesis were investigated by high field, high frequency (95 GHz) EPR. Measurements on frozen solutions of D + , a dimeric π-cation radical, of various organisms ( Rps. viridis, Rb. sphaeroides, Chloroflexus aurantiacus and a heterodimer mutant) are reported, revealing differences in the principal values of the G -tensor. Elements of a theory relating the magnitudes of G principal values to the electronic structure are discussed.

Liquid solution ENDOR and TRIPLE resonance experiments have been performed on texaphyrin radical anions generated by sodium reduction in tetrahydrofuran. Six proton and two nitrogen hyperfine couplings could be determined, including their signs. The results were compared with theoretical isotropic hyperfine couplings. They were calculated for energy-minimized texaphyrin structures, including the sodium metallo-derivative, by state-of-the-art SCF-MO methods (RHF-INDO/SP). This comparison suggests that in the course of the alkali metal reduction of texaphyrin the acidic hydrogen in the macrocycle is replaced by sodium.

Single crystals of 17α,21-dihydroxyprogesterone were γ-irradiated at 295 K and studied using X-band ESR and ENDOR. Two types of radical were observed and a structure was assigned to the specimen by analysing the orientational variation of the spectra. The first radical is formed by abstraction of the hydrogen atom from the C(6) carbon while the unpaired electron is delocalized onto the system O(3) C(3) C(4) C(5) C(6). The second radical is formed by attachment of the hydrogen atom to the oxygen O(3) of the carbonyl group, and delocalization of the unpaired electron onto the atoms C(3), C(4) and C(5). The effect of substitution of hydroxyl groups on the hyperfine splitting tensor has been also analysed.

The electron spin density distribution of the cation radical of the primary donor, D+, a bacteriochlorophyll a dimer was determined by ENDOR and TRIPLE resonance experiments performed on single crystals of reaction centers (RCs) of Rhodobacter sphaeroides R-26. Nine isotropic proton hyperfine coupling constants (hfc's) were obtained and from the angular dependence of the hfc's in three crystallographic planes, five complete hyperfine (hf) tensors were determined. Theoretical hf tensors were calculated by the all-valence-electron SCF molecular orbital method RHF-INDO/SP using the X-ray structure data of the dimer D and its amino acid environment. A comparison of the directions of the principal axes of the experimental and calculated hf tensors enabled us to identify the hfc's with specific protons on the two bacteriochlorophyll halves DL and DM of the dimer. The result shows that the unpaired valence electron is unequally distributed over the dimer halves, favoring DL by approx. 2:1. This ratio has been obtained from the proton hfc's of rotating methyl groups, which directly reflect the π-spin densities at the corresponding positions in the two macrocycles, DL and DM. It was further confirmed by recent 15N-ENDOR experiments on RC single crystals (Lendzian, F., Bonigk, B., Plato, M., Mobius, K. and Lubitz, W. (1992) in The Photosynthetic Bacterial Reaction Center II (Breton, J. and Vermeglio, A., eds.), pp. 89–97, Plenum Press, New York). The observed asymmetry of D+ is attributed to the difference in energies of the highest filled molecular π-orbitals of the monomeric halves, DL and DM, which is caused by differences in the structure of the two bacteriochlorophylls and/or their environment. Possible implications of this asymmetry for the electron transfer in the RC are discussed.

The triplet states of several substituted porphyrins (Tetraphenylporphyrin (H2TPP), Zinc-Tetramethylporphyrin (ZnTMP), Octaethylporphyrin (H2OEP) and the Dication of H2TPP (H4TPP2+)) and two covalently linked dimers with H2TPP-subunits in disordered solid solution were studied by EPR and ENDOR at liquid helium temperature. The measurement yields theAzz component of the hyperfine tensors of all α-protons in the reference frame of the zero field splitting tensor. Dipolar and isotropic contributions toAzz are discussed and spin densities derived. The spin densities are compared with results of all-valence-electrons self-consistent field molecular orbital calculations (RHF-INDO/S). One of the dimers shows indications of triplet energy transfer between the porphyrin subunits. The order of magnitude of the transfer rate is estimated to be 5 · 105 s−1.

Porphycene is a structural isomer of porphyrin. The photoexcited triplet states of porphycene, 2,7,12,17-tetra-n-propylporphycene and 9,10,19,20-tetra-n-propylporphycene in disordered solid solution were studied by EPR and ENDOR. The ENDOR spectra yield the hyperfine tensor elementsA zz for each of the different groups of equivalent protons. The dipolar contribution toA zz is estimated and spin densities are derived from the isotropic contribution. They are compared with results of all-valence-electrons self-consistent field molecular orbital calculations (RHF-INDO/SP and RHF-INDO/S).

Intramolecular electron transfer (ET) in cis and trans isomers of a covalently linked porphyrin–cyclohexylene–quinone, oriented in liquid crystals (LCs), is monitored by time‐domain electron paramagnetic resonance (EPR) spectroscopy over an extended range of temperature, i.e., 210

Light-induced triplet electron transfer (ET) is observed in two isomeric cyclohexene-bridged porphyrin–quinones (P–Q) in highly viscous ethanol at temperatures between 130 and 150 K. Both the primary porphyrin triplet, TP–Q, and the triplet radical pair, T(P·+–Q·–), identified by their dipolar splittings and polarization patterns, are detected by time-resolved continuous-wave electron paramagnetic resonance (cw-EPR). A theoretical model is presented by which the kinetic traces at the turning points of the powder-type EPR spectrum of T(P·+–Q·–) are simulated. From the fits to the experimental data, values for the ET rate, kET, and the exchange integral, J, are estimated. At 150 K, for example, kET= 0.26 × 106 s–1(±0.02 × 106 s–1) and J=–1370 G (±60 G). The rates obtained for the charge recombination from the singlet radical pair to the ground state indicate that a distribution of J values has to be considered, even for P–Qs with a relatively rigid bridge.

The directionality of light-induced charge transfer in bacterial photosynthetic reaction centers (RCs) with respect to their A and B cofactor branches is still poorly understood on the electronic level. A prominent example is primary electron transfer in the RCs from the purple bacterium Rb. sphaeroides. Site-directed mutants with specific alterations of the cofactor binding sites with respect to the native system can deliver useful information toward a better understanding of the directionality enigma. Here we report on electron paramagnetic resonance (EPR) studies of the LDHW quadruple mutant, HL(M182)/GD(M203)/LH(M214)/AW(M260), which contains crucial mutations in the electron-transfer pathway. The directionality of the charge separation process was studied under light- or dark-freezing conditions first directly by 95 GHz (W-band) high-field EPR spectroscopy examining the charge-separated radical pairs (P865•+ Q(B)•−) of the primary donor P865, a bacteriochlorophyll dimer, and the terminal acceptor, QB, a ubiquinone-10. Second, it was studied indirectly by 34 GHz (Q-band) EPR examining the triplet states of the primary donor ((3)P865) that occur as a byproduct of the photoreaction. At 10 K, the triplet state has been found to derive mainly from an intersystem crossing mechanism, indicating the absence of charge-separated radical-pair states with a lifetime longer than 10 ns. B-branch charge separation and formation of the triplet-state (3)P865 via a radical-pair mechanism can be induced with low yield at 10 K by direct excitation of the bacteriopheophytins in the B-branch at 537 nm. At this wavelength, charge separation most probably proceeds via hole transfer from bacteriopheophytin to the primary donor. The triplet state of the primary donor is found to be quenched by the carotenoid cofactor present in the RC. The light-induced transient EPR signal of P•+ Q(B)•− is formed in a minor fraction of RCs (1%) for RCs frozen in the dark. In contrast, about 70% of RCs illuminated upon freezing are trapped in the long-lived (τ104 s) charge-separated-state P•+ Q(B)•−. The temperature dependence of the EPR signals from P•+ Q(B)•− points to two factors responsible for the forward electron transfer to the terminal acceptor QB and for the charge-recombination reaction. The first factor involves a significant protein conformational change to initiate P•+ Q(B)•− charge separation, presumably by moving the quinone from the distal to the proximal position relative to the iron. The second factor includes protein relaxation, which governs the charge-recombination process along the B-branch pathway of the LDHW mutant.

The oxidized primary electron donor P865+ in reaction center single crystals of Rb. sphaeroides wild type 2.4.1 has been investigated by electron-nuclear-nuclear TRIPLE resonance techniques. The measured anisotropic and isotropic hyperfine couplings have been compared with data from the carotenoidless mutant R-26 of the same organism. There is no detectable structural difference of the dimer P865+ in both strains and its orientation in the crystal unit cell axes is the same within ±3°. However, a small shift of spin density towards the L-half of the dimer is observed due to the presence of the carotenoid.

The EPR g -factor rotation pattern of the primary donor cation radical in the x  y plane of illuminated reaction center single crystals of Rb. sphaeroides R-26 near room temperature was measured at 3 mm wavelength (W-band, 95 GHz). In contrast to earlier X-band (9.5 GHz) EPR experiments, the two magnetically inequivalent sites of the four reaction centers per unit cell were well resolved. in frozen reaction center solution was also measured by 3 mm EPR. The analysis of both the frozen-solution spectrum and the single-crystal rotation pattern yields the principal values of the g -tensor and the phase angle between the two magnetically inequivalent sites in the x  y plane.

Distance and relative orientation of functional groups within protein domains and their changes during chemical reactions determine the efficiency of biological processes. In this work on disordered solid-state electron-transfer proteins, it is demonstrated that the combination of pulsed high-field EPR spectroscopy at the W band (95 GHz, 3.4 T) with its extensions to PELDOR (pulsed electron-electron double resonance) and RIDME (relaxation-induced dipolar modulation enhancement) offers a powerful tool for obtaining not only information on the electronic structure of the redox partners but also on the three-dimensional structure of radical-pair systems with large interspin distances (up to about 5 nm). Strategies are discussed both in terms of data collection and data analysis to extract unique solutions for the full radical-pair structure with only a minimum of additional independent structural information. By this novel approach, the three-dimensional structure of laser-flash-induced transient radical pairs P(865)(*+)Q(A)(*-) in frozen-solution reaction centers (RCs) from the photosynthetic bacterium Rhodobacter (Rb.) sphaeroides is solved. The measured positions and relative orientations of the weakly coupled ion radicals P(865)(*+) and Q(A)(*-) are compared with those of the precursor cofactors P865 and QA known from X-ray crystallography. A small but significant reorientation of the reduced ubiquinone QA is revealed and interpreted as being due to the photosynthetic electron transfer. In contrast to the large conformational change of Q(B)(*-) upon light illumination of the RCs, the small light-induced reorientation of Q(A)(*-) had escaped previous attempts to detect structural changes of photosynthetic cofactors upon charge separation. Although small, they still may be of functional importance for optimizing the electronic coupling of the redox partners in bacterial photosynthesis both for the charge-separation and charge-recombination processes.

Ultraviolet radiation promotes the formation of a cyclobutane ring between adjacent pyrimidine residues on the same DNA strand to form a pyrimidine dimer. Such dimers may be restored to their monomeric forms through the action of a light-absorbing enzyme named DNA photolyase. The redox-active cofactor involved in the light-induced electron transfer reactions of DNA repair and enzyme photoactivation is a noncovalently bound FAD. In this paper, the FAD cofactor of Escherichia coli DNA photolyase was characterized as the neutral flavin semiquinone by EPR spectroscopy at 9.68 and 94.5 GHz. From the high-frequency/high-field EPR spectrum, the principal values of the axially symmetric g-matrix of FADH(*) were extracted. Both EPR spectra show an emerging hyperfine splitting of 0.85 mT that could be assigned to the isotropic hyperfine coupling constant (hfc) of the proton at N(5). To obtain more information about the electron spin density distribution ENDOR and TRIPLE resonance spectroscopies were applied. All major proton hfc's could be measured and unambiguously assigned to molecular positions at the isoalloxazin moiety of FAD. The isotropic hfc's of the protons at C(8alpha) and C(6) are among the smallest values reported for protein-bound neutral flavin semiquinones so far, suggesting a highly restricted delocalization of the unpaired electron spin on the isoalloxazin moiety. Two further hfc's have been detected and assigned to the inequivalent protons at C(1'). Some conclusions about the geometrical arrangement of the ribityl side chain with respect to the isoalloxazin ring could be drawn: Assuming tetrahedral angles at C(1') the dihedral angle between the C(1')-C(2') bond and the 2p(z)() orbital at N(10) has been estimated to be 170.4 degrees +/- 1 degrees.

For an efficient charge separation in quinones coupled covalently with porphyrin, the MO coefficient in the HOMO of the porphyrin at the site of coupling should be as small as possible and the orientation of the quinone to the cyclohexane bridge as orthogonal as possible

The molecular structure of free radicals formed in γ-irradiated orthorhombic single crystals of hydrated testosterone was investigated by Electron Nuclear Double Resonance (ENDOR) spectroscopy. Only one kind of radical was observed, which is formed by addition of hydrogen atom to oxygen atom O(3). We observed interaction of the unpaired electron, which is delocalized on the carbons C(3), C(4) and C95), with one α-proton in position 4 and with four unequivalent β-protons connected with the carbon atoms C(2) and C(6). The matrices of the hyperfine couplings and the g -factor of the radical are given.

Recent Electron-Nuclear-Double and -Triple Magnetic Resonance measurements on single crystals of reaction centers (RC’s) of the bacterium Rb. sphaeroides R-26 have revealed an asymmetric spin density distribution of the primary donor radical cation P 865 + 1. The observed ratio of spin densities, ρ L/ρ M, on the monomeric halves DL and DM of this dimer is approximately 2 : 1. A similar result had been found earlier for the primary donor radical cation P 960 +. in RC’s of Rps. viridis in liquid solution 2,3.

The rates of the electron transfer processes in reaction centers (RC’s) of photosynthetic bacteria are controlled both by the spatial and the electronic structure of the involved donor and acceptor molecules. The spatial structure of bacterial RC's has been determined by X-ray diffraction for Rhodopseudomonas (Rp.) viridis 1 and for Rhodobacter (Rb.) sphaeroides.2 The electronic structure of the transient radical species formed in the charge separation process can be elucidated by EPR and ENDOR techniques.3 The information is contained in the electron-nuclear hyperfine couplings (hfc’s) which, after assignment to specific nuclei, yield a detailed picture of the valence electron spin density distribution in the respective molecules.3,4

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

Spin density calculations were performed on the oxidized primary electron donor in reaction centers (RC’s) from Rhodobacter sphaeroides R-26. The calculations were based on the all-valence-electron MO method RHF-INDO/SP and on the three-dimensional structure determined by X-ray diffraction. Results are compared with experimental isotropic hyperfine couplings (hf c’s) obtained from ENDOR/TRIPLE magnetic resonance spectra and with previous studies on in RC’s from Rhodopseudomonas viridis. Both bacteriochlorophyll dimer species and show marked deviations of their spin distributions from C2-symmetry. However, is more symmetric than with respect to sums of spin densities on the A and B halves of the dimers. The effects of various environmental (inclusion of surrounding amino acid residues) and structural refinements (e.g. rotation of acetyl groups) on hfc’s and excited state properties were studied. The latter were obtained by additional INDO/S calculations. In both species, P865 and P960, the excited electron in the lowest unoccupied molecular orbital (LUMO) is predicted to be preferentially on the B half of the dimer, which is close to the accessory bacteriochlorophyll monomer on the photoactive A branch. Thus the preferential electron transfer along the A branch originates in the excited state of the dimer.

Anisotropic hyperfine interactions of P 865 +· are obtained from ENDO experiments by rotation about the C2 axes of reaction center (RC) single crystals of Rb. Sphaeroids R-26. An assignment to individual molecular positions in P 865 +· is aqchieved by comparison with calculated dipolar HFS tensors using atomic coordinates from x-ray diffraction.

In the ENDOR spectra of some silacyclopentadiene anion radicals in solution, temperature-dependent line widths and line positions were observed. This dynamic behaviour could be interpreted as being due to hindered rotation of substituted phenyl rings. It was possible to determine activation energies and entropies, from which it is concluded that the main part of the potential barrier stems from π conjugation effects. The observed dynamic effects were also exploited for assignment of ENDOR lines to molecular positions.

Solution 25 Mg ENDOR and TRIPLE resonance experiments have been performed on the bacteriochlorophyll a (BChl a) anion and cation radicals enriched in 25 Mg. The 25 Mg isotropic hyperfine coupling constants for BChl a + · and BChl a − · have been determined. The values are in good agreement with those obtained from recent MO calculations.

From chemically generated anion radicals of anti[2.2](1,4)naphthalenophane and 1,4-dimethylnaphthalene, hyperfine coupling constants including relative signs are determined by ENDOR and TRIPLE resonance. The data are compared with spin density distributions obtained by MO calculations. The results are discussed with respect to the spin density distribution in the “special pair” primary donor of bacterial photosynthesis.

The radical cation of bacterioehlorophyll b (BChl b) is investigated by ENDOR and TRIPLE resonance in liquid solution. The experimental hyperfine coupling constants, ten proton and three nitrogen couplings, are compared with the predictions from advanced molecular-orbital calculations (RHF INDO/SP). The detailed picture obtained of the spin density distribution is a prerequisite for the investigation of the primary electron donor radical cation in BChl b containing photosynthetic bacteria.

Distant ENDOR was used as a sensitive spectroscopic method for the determination of molecular structure parameters of C2H4(COOD)2 and C2D4(COOH)2 in their diamagnetic singlet ground state. The structural information was obtained by measuring the quadrupole coupling tensors of the deuterium atoms in the CD2 positions and in the intermolecular hydrogen bonds OD … O and by measuring the dipolar coupling tensors between the protons in the CH2 group and between the carboxylic protons in adjacent hydrogen bonds. The distant ENDOR results agree very well with crystallographic data.

Two representative porphyrin dimers, each containing meso-tetraphenylporphyrin (TPP) monomers as constituents, have been studied by resonance Raman (RR) spectroscopy. In both cases the monomer units are linked through one common phenyl ring. The first incorporates a para-phenylene bridge thereby retaining a centre of inversion; the second incorporates a metaphenylene bridge and has no centre of inversion. Modes giving rise to distinguishing features (monomer versus dimer and the p -dimer versus the m -dimer) in the 300–420 cm −1 region are qualitatively assigned. It is concluded that in each dimer the componentmonomers are weakly interacting in their Soret excited states. The excitation may be considered monomer-localised on the RR time scale of ≈ 1 ps.

From the ENDOR spectra of the mononegative ions of biphenyl and ortho-, meta- and para-terphenyl in solution complete sets of hyperfine splitting constants could be determined. The splittings are interpreted in terms of SCF MO spin densities obtained from McLachlan type calculations. It turns out that π-σ spin delocalization effects do not play a significant role in the spin density distribution in these radicals although some of them deviate considerably from coplanarity.

In the liquid crystal “Phase IV” which exhibits its nematic range between 16 and 76 °C ENDOR spectra of perinaphthenyl and tri- t -butylphenoxyl radicals could be recorded. It is demonstrated that the combined use of the ENDOR and liquid crystal methods yeilds valuable structural information.

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

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

The structure of the primary donor P in bacterial photosynthesis is of considerable interest due to the unique role this species plays in the very first events of the charge-separation process in the reaction center (RC). It could be shown that P is a specialized bacteriochlorophyll (BChl) [l] which was proposed by NORRIS et al. [2] to be a BChl-dimer. This “special pair” model was a matter of considerable controversy in recent years. Different dimer models [3, 4, 5] and perturbed monomer models [6, 7] have been proposed for the primary donor. The existence of a dimer has recently been demonstrated, at least for Rhodopseudomonas (Rp.) viridis RC’s by an X-ray structure analysis of RC single crystals [8]. An alternative approach to the characterization of the primary donor in its cationic state is provided by EPR and electron nuclear doubleresonance (ENDOR) spectroscopy [4, 5, 9–12]. In this paper we summarize the most recent contributions of EPR and ENDOR in elucidating the electronic structure details of the primary donor cation radicals. In particular P+.865in RC’s of Rp. sphaeroides and Rhodospirillum (R.) rubrum, and P+.960 in RC’s of Rp. viridis are studied. The isotropic hyperfine coupling constants (hfc’s) extracted by ENDOR and TRIPLE resonance [13, 14] are compared with values from advanced MO calculations, thereby relating the experimental values to structural details of the dimers.

The light-induced cation radical of the primary electron donor, P 870 +· , in photosynthetic reaction centers from Rhodospirillum rubrum G-9, has been investigated by electron-nuclear double resonance (ENDOR) in liquid aqueous solution. The measured hyperfine coupling constants are assigned to specific molecular positions by partial deuteration. Comparison with the bacteriochlorophyll a cation radical shows different reduction factors of the individual coupling constants deviating from the value 2.0 reported in earlier investigations in frozen solutions. The average of the coupling constants is, however, reduced by a factor very close to 2.0. EPR simulations using the ENDOR coupling constants support a dimer model for P 870 +· with C 2 symmetry, where the two macrocycles are close enough to form a supermolecular orbital resulting in a different distribution of the unpaired electron, compared with the monomeric bacteriochlorophyll a cation radical. Molecular orbital calculations were used to obtain structural information about this dimer.

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