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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.

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.

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.

EPR spectroscopy at high frequencies and low temperatures and ENDOR spectroscopy at X- and W-bands have been applied to investigate two- and three-spin complexes of the type MQ2˙ and MQ3˙ generated by mechanochemical treatment (by grinding or by the action of elastic waves) of solid mixtures of quinones and metals (M = Al, Ga, In, Cd, Sn, Zn). In addition, complexes obtained in liquid-phase reactions of metal amalgams with quinones were investigated. The magnetic-resonance parameters and the observed thermal spin polarization provide evidence for the identity of the magnetic complexes produced in solutions and by solid-state mechanochemical treatment. In particular, the presence of ground-state triplet and quartet species has been demonstrated in both cases. In addition, the exchange and the dipole–dipole interaction parameters were evaluated, their comparison providing information about the electronic intramolecular interaction.

Light-induced triplet electron transfer (ET) and subsequent triplet radical-pair (RP) recombination in two covalently linked porphyrin–quinone systems in highly viscous ethanol has been investigated by both transient optical absorption and time-resolved resonance Raman spectroscopy with a time resolution of 10 ns. The temperature dependence of the rates is measured between 155 and 200 K and compared with predictions of solvent-controlled adiabatic electron-transfer theory. It is shown that the triplet ET in the normal region (exergonicity ΔG0 λs) depends on the dynamics of the exchange interaction J, on the triplet–singlet mixing of the radical pair states and on the singlet recombination rate. An intermolecular ET process leading to a disproportionation reaction of the quinone moieties is also observed.

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 spin density distributions in the cation radicals of various covalently linked tetraphenyl porphyrin dimers and of their monomeric constituents have been studied by liquid-phase EPR, ENDOR and TRIPLE resonance methods to find out whether the dimer systems show intramolecular electron delocalization. Such delocalization is known to occur in the bacteriochlorophyll dimer (‘special pair’) in photosynthetic reaction centres. The dimers studied in this work are derived from zinc meso-tetraphenylporphyrin (ZnTPP) moieties linked either by a phenyl ring at para and meta positions or by a —CH2— bridge at para positions. 1H and 14N hyperfine coupling constants were measured for the dimer and ZnTPP cation radicals. By comparing the hyperfine data it was concluded that in the phenyl-bridged porphyrin dimers the unpaired electron is delocalized over the dimer halves (similar to the ‘special pair’) whereas in the CH2-bridged dimer the unpaired electron is localized on one porphyrin unit.

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.

The detection of hyperfine couplings in large and low-symmetry molecules is often hampered by problems of sensitivity and/or resolution. In such situations solutions to the problem are offered by simultaneously driving n.m.r., e.s.r. and optical transitions leading to well characterized electron–nuclear double and triple resonance (ENDOR, TRIPLE) and optically detected quadrupole resonance (ODNQR) spectra. The basic strategies of these multiple resonance techniques are discussed, together with applications to photosynthetic systems and spin-polarized aromatic molecules.