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 P₇₀₀ and the secondary electron acceptor A₁ of Photosystem I (PSI) using 130 GHz (D-band) EPR and Electron–Nuclear-Double-Resonance (ENDOR) spectroscopy. PSI was isotopically labeled with ¹⁵N (I = ½) to avoid quadrupolar interactions in the most abundant nitrogen isotope ¹⁴N (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 P₇₀₀ and the involvement of protein nitrogen atoms with reduced A₁. While HF ENDOR of A₁⁻ allows identification of two nitrogen atoms, HF ENDOR of P₇₀₀⁺ still does not permit unique assignment of the recorded hyperfine couplings.

数十亿年来，大自然优化了将光能转化为化学能的光合作用机制。光合作用的关键初级反应发生在大的膜蛋白-辅因子复合物中。光诱导的顺序电子转移反应通过嵌入蛋白质基质中的供体/受体辅因子链发生，从而导致长寿命的跨膜电荷分离状态。EPR 是研究电子转移和蛋白质环境与氧化还原活性辅因子相互作用的首选方法。然而，有机辅因子自由基的光谱通常不能完全解析，并且在传统的 X 波段 EPR 中严重重叠。即使在 Q 波段 EPR 中，这种重叠也是存在的，而且通常是一个严重的问题。其结果，报告的 EPR 数据存在很大差异，并且对几种氧化还原活性辅因子的电子结构的了解有限。这些严重的问题通常可以通过高频 EPR (HF EPR) 提供的出色光谱分辨率来克服。在这里，我们研究了初级电子供体 P 的电子结构₇₀₀和光系统 I (PSI)的二次电子受体 A ₁使用 130 GHz（D 波段）EPR 和电子核双共振 (ENDOR) 光谱。PSI 用¹⁵ N ( I  = ½)同位素标记，以避免最丰富的氮同位素¹⁴ N ( I  = 1)中的四极相互作用并简化 ENDOR 光谱。ENDOR 光谱对于确定包含氧化 P ₇₀₀的两个叶绿素分子的氮原子的超精细耦合以及蛋白质氮原子与还原 A ₁的参与至关重要。虽然 A ₁ ^{− 的}HF ENDOR允许识别两个氮原子，但 P 的 HF ENDOR₇₀₀ ⁺仍然不允许对记录的超精细耦合进行唯一分配。