Authors

Abstract

For billions of years, nature has optimized the photosynthetic machinery that converts light energy into chemical energy. Key primary reactions of photosynthesis occur in large membrane protein-cofactor complexes. The light-induced sequential electron transfer reactions occur through a chain of donor/acceptor cofactors embedded in the protein matrix resulting in a long-lived transmembrane charge-separated state. EPR is the method of choice to study electron transfer and the interaction of protein environment with redox-active cofactors. However, the spectra of organic cofactor radicals typically are not fully resolved and severely overlap at conventional X-band EPR. Even at Q-band EPR, this overlap is present and often a serious problem. As a result, there is a large variation of the reported EPR data and limited understanding of electronic structures of several redox-active cofactors. These serious problems can often be overcome by the excellent spectral resolution provided by high-frequency EPR (HF EPR). Here, we study the electronic structure of the primary electron donor P-700 and the secondary electron acceptor A(1) of Photosystem I (PSI) using 130 GHz (D-band) EPR and Electron-Nuclear-Double-Resonance (ENDOR) spectroscopy. PSI was isotopically labeled with N-15 (I = 1/2) to avoid quadrupolar interactions in the most abundant nitrogen isotope N-14 (I = 1) and simplify the ENDOR spectra. ENDOR spectroscopy is central for determining the hyperfine coupling of nitrogen atoms of the two chlorophyll molecules comprising oxidized P-700 and the involvement of protein nitrogen atoms with reduced A(1). While HF ENDOR of A(1)(-) allows identification of two nitrogen atoms, HF ENDOR of P-700(+) still does not permit unique assignment of the recorded hyperfine couplings.