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Over the last 10 to 20 years NMR spectroscopy has advanced tremendously by employing sophisticated pulsed radio-frequency irradiation schemes and by exposing the sample to the strong magnetic fields attainable in superconducting magnets. Pulsing the rf field led to Fourier transform and two-dimensional correlation NMR; increasing B 0 and the rf frequency led to improved spectral resolution and sensitivity. Both developments were of decisive importance for successful applications of NMR to biological systems (Ernst et al., 1987; Wuthrich, 1986).

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

In molecular spectroscopy, the introduction of more than one resonant electromagnetic field is a well-established technique applied in the full spectral range between radiofrequency (rf) and ultraviolet.(1) The main motivation for extending single resonance to double or multiple resonance techniques can be twofold: (1) either one wants to increase the sensitivity of detection by “quantum transformation” from low-frequency absorbed quanta to high-frequency detected quanta, or (2) one wants to enhance the resolution of the spectra, i.e., reduce the number of spectral lines in a given frequency range by introducing specific coherent time-dependent interactions that average out unwanted static interactions in the Hamiltonian,(2) by imposing additional “selection rules,”(2) or by exploiting degeneracies of certain transitions.(3)

For magnetic resonance studies of paramagnetic species electron paramagnetic resonance (EPR) is a well-established method. However, when trying to elucidate the electronic structure of large and lowsymmetry radicals, as they typically occur in biological systems, one is often hampered by problems of spectral resolution. It was as early as 1956 when Feher (1956) demonstrated that by electron nuclear double resonance (ENDOR) the spectral resolution can be greatly improved. ENDOR signals are obtained by monitoring the changes of the amplitude of a saturated EPR line that occur when sweeping the frequency of an additionally applied rf field through the nuclear (NMR) region. This first ENDOR experiment was technically feasible only because the sample—phosphorus doped silicon—was studied at low temperature, where all the relaxation times are sufficiently long to easily obtain saturation. For radicals in liquid solution, however, these relaxation times are much shorter—on the order of 10-5-10-7 sec—and, consequently, ENDOR-msolution experiments are technically much more sophisticated since much larger saturating microwave and rf fields have to be applied. This probably explains why the first ENDOR-in-solution experiments required many more years before they could be successfully performed by Cederquist (1963) and by Hyde and Maki (1964).

One outstanding question in bacterial photosynthesis research concerns the relevance of the dimeric nature of the primary donor P for the fast electron transfer step from the first excited singlet state 1P*H to the charge separated state P+H−, where H stands for the intermediate acceptor bacteriopheophytin. This problem can only be attacked by performing a detailed study of the electronic structure of the primary donor itself and of the electronic interactions between all involved cofactors in their various active states. This requires the combined effort of high resolution X-ray crystallography, powerful spectroscopic methods and sufficiently advanced quantum-chemical calculations.