Determining the Electronic Structure of Paramagnetic Intermediates in membrane proteins: A high-resolution 2D ¹H hyperfine sublevel correlation study of the redox-active tyrosines of photosystem II

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.