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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.

In der aktuellen Photosyntheseforschung interessieren vor allem die Elektronentransferreaktionen der Ladungstrennung und -rekombinationen, die unmittelbar nach Lichteinfall ablaufen. Ein Elektron springt dabei eine Kette von Farbstoffmolekulen entlang, die in einer Protein-matrix eingebettet sind, und liefert die elektrochemische Energie fur die nachgeschalteten chemischen Dunkelreaktionen zur CO2-Assimilation. Zur Aufklarung der Elektronentransfer-Schritte werden mit Erfolg Elektronenspin-Resonanz (EPR)-Experimente durchgefuhrt, in denen nach einem kurzen Laserblitz die Strukturen und dynamischen Eigenschaften der am Ladungstransfer beteiligten Molekulzustande gemessen werden. Das in Berlin von den drei Autoren entwickelte gepulste Hochfeld-EPR-Spektrometer arbeitet bei zehnfach hoheren Mikrowellenfrequenzen und Magnetfeldern als bisherige kommerzielle Gerate. Dadurch wird es moglich, an winzigen Probenmengen auf wenige milliardstel Sekunden genau die lichtinduzierten Reaktionen zu untersuchen und die Struktur der Reaktionspartner zu bestimmen, was fur ein Verstandnis komplexer biophysikalischer Prozesse unerlaslich ist. In Deutschland ist dieses Hochfeld-Gerat bisher einzigartig.

Time-resolved EPR spectra are reported for photoexcited porphyrin—quinones with butylene and cis- or trans- cyclohexylene spacers between porphyrin donors and crown ether—quinone acceptors. In the nematic phases of liquid crystals, polarized triplet EPR spectra of the porphyrin moiety could be observed at early delay times (0.1–0.6 μs) after laser flash irradiation. At later delay times (1–2 μs) the triplet EPR spectra of the charge-separated radical pair state, created by intramolecular electron transfer from the porphyrin donor to the crown ether quinone acceptor, could be detected. The similarity of the zero-field splitting parameters D of the cyclohexylene- and butylene-linked compounds shows that the radical pair of the flexible butylene-linked compound is in a stretched conformation. However, longer laser irradiation of the species with the flexible bridge results in the formation of a novel triplet radical pair species with a shorter porphyrin—quinone separation indicating a photoinduced backfolding of the quinone over the porphyrin plane. Comparison of the cis- and trans-cyclohexylene-linked compounds reveals a distortion of the cyclohexylene spacer in the cis compound.

In this overview, modern multifrequency EPR spectroscopy, in particular at high magnetic fields, is shown to provide detailed information about structure, motional dynamics, and spin chemistry of transient radicals and radical pairs occurring in photochemical reactions. Examples discussed comprise photochemical reactions in liquid solution and light-initiated electron transfer processes both in biomimetic donor–acceptor model systems in frozen solution or liquid crystals and in natural photosynthetic-reaction-center protein complexes. The transient paramagnetic states exhibit characteristic electron polarization (CIDEP) effects. They contain valuable information about structure and dynamics of the transient reaction intermediates. Moreover, they are exploited for signal enhancement. Continuous-wave (cw) and pulsed versions of time-resolved high-field EPR spectroscopy, such as cw-transient-EPR (TREPR) and pulsed-electron-spin-echo (ESE) experiments, are compared with respect to their advantages and limitations for the specific system under study. For example, W-band (95-GHz) TREPR spectroscopy in conjunction with a continuous-flow system for light-generated short-lived transient spin-polarized radicals of organic photoinitiators in solution was performed with a time resolution of 10 ns. The increased Boltzmann polarization at high fields even allows detection of transient radicals without CIDEP effects. This enables one to determine initial radical polarization contributions as well as radical-addition reaction constants. Another example of the power of combined X-band and W-band TREPR spectroscopy is given for the complex electron-transfer and spin dynamics of covalently linked porphyrin–quinone as well as Watson–Crick base-paired porphyrin–dinitrobenzene donor–acceptor biomimetic model systems. Furthermore, W-band ESE experiments on the spin-correlated coupled radical pair in reaction centers of the purple photosynthetic bacterium Rb. sphaeroides reveal details of distance and orientation of the pair partners in their charge-separated transient state. The results are compared with those of the ground-state P865QA. The high orientation selectivity of high-field EPR provides single-crystal-like information even from disordered frozen-solution samples. The examples given demonstrate that high-field EPR adds substantially to the capability of ‘classical’ spectroscopic and diffraction techniques for determining structure–dynamics–function relations of biochemical systems, since transient intermediates can be observed in real time in their working states on biologically relevant time scales.

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 physical principles underlying steady-state electron-nuclear double resonance (ENDOR) and electron-nuclear-nuclear triple resonance (TRIPLE) experiments on radicals in solution are discussed. The gain in spectral resolution achievable by these techniques, as compared with standard ESR, allows hyperfine coupling constants to be measured even for large and low-symmetry radicals. ENDOR/TRIPLE is applied to cation radicals related to bacterial photosynthesis. The measured hyperfine couplings are interpreted by means of the all-valence electron MO method RHF-INDO/SP. Information about the spatial structure of chromophores in the reaction centers of photosynthetic bacteria, Rb. sphaeroides and Rps. viridis, is obtained from an analysis of the spin density distribution and from minimization of the total energy. The results are compared with recently available structural data of the primary donor obtained from X-ray diffraction experiments. The spin density distribution in the primary donor cation radical P+960, which consists of two bacteriochlorophyll b molecules, is discussed in terms of symmetry and perturbations from the protein environment.

The light-induced radical cation of the primary electron donor, P+.960, in photosynthetic reaction centers (RCs) from Rhodopseudomonas viridis has been investigated by various magnetic resonance techniques (ESR, ENDOR, TRIPLE). Comparison of the measured hyperfine couplings with those of the cation radical of monomeric bacteriochlorophyll b (BChl b) and with RHF-INDO/SP molecular orbital calculations, performed on P+.960 using coordinates of an X-ray structure analysis, consistently show an asymmetric distribution of the unpaired electron over the two BChl b molecules which constitute P960. The derived spin density ratio is 2.8 in favor of the dimer half bound to the L protein subunit. The possible relevance of this result for the primary electron transfer step in the RC is briefly discussed.

The spin density distributions in the cation radicals of various covalently linked porphyrin dimers have been studied by liquid phase ESR and ENDOR methods to find out whether these systems show intramolecular electron delocalization. Such a delocalization is known to occur in the bacteriochlorophyll dimer (“special pair”) in the photosynthetic reaction center. The dimers that were studied in this work were derived from zinc mesotetratolylporphyrin (ZnTTP) and linked at the ortho or para positions of one phenyl ring with varying bridge lengths. 1H and I4N hyperfine coupling constants could be measured for the dimer cation radicals and compared with those of monomeric ZnTTP as well as ZnTTP derivatives that carry alkoxy or hydroxy substituents to mimic the bridges of the dimers. By comparing the hyperfine data of the monomer and dimer cation radicals it is concluded that in the present dimers the unpaired electron is localized on one porphyrin unit.