The photosynthetic reaction center, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in nature by using light energy to drive water oxidation. The four-electron water oxidation reaction occurs at the tetranuclear manganese‑calcium-oxo (Mn₄Ca-oxo) cluster that is present in the oxygen-evolving complex (OEC) of PSII. The water oxidation reaction is facilitated by proton-coupled electron transfer (PCET) at the redox-active tyrosine residue, Y_Z, in the OEC which is one of the two symmetric tyrosine residues, Y_Z and Y_D, in PSII. Although Y_Z and Y_D are chemically identical, their redox properties and reaction kinetics are very different. In the present study, we apply high-resolution two-dimensional (2D) ¹H hyperfine sublevel correlation (HYSCORE) spectroscopy to determine the electronic structure of Y_Z and Y_D to understand better the functional tuning of PCET at each tyrosine. Most importantly, the 2D HYSCORE measurements that are described here are applicable for the study of paramagnetic cofactors in a wide variety of membrane-bound proteins.

光合作用反应中心光系统II（PSII）通过使用光能驱动水氧化来催化自然界中最耗能的反应之一。四电子水氧化反应发生在PSII的析氧复合物（OEC）中存在的四核锰-钙-氧代（Mn ₄ Ca-氧代）簇上。通过在OEC中的氧化还原活性酪氨酸残基Y _Z上的质子耦合电子转移（PCET）促进水氧化反应，该OEC是PSII中两个对称的酪氨酸残基Y _Z和Y _D之一。虽然Y _Z和Y _D在化学上是相同的，它们的氧化还原特性和反应动力学有很大不同。在本研究中，我们应用高分辨率二维（2D）¹ H超细亚级相关（HYSCORE）光谱确定Y _Z和Y _D的电子结构，以更好地了解每个酪氨酸上PCET的功能调节。最重要的是，此处描述的2D HYSCORE测量可用于研究多种膜结合蛋白中的顺磁性辅因子。