A study of the exchangeable protons in the QA site of photosystem II
Authors: Garmany Armin , Runyon Zachary David , Kinzer David Stephen , Dikanov Sergei , Kolling Derrick R.J. 
Published: November 2023

Plants have developed the only biological system capable of oxidizing water. Through the oxidation of water plants are able to convert light energy into chemical energy. This water oxidation is catalyzed by photosystem II (PSII) at a site called the oxygen‐evolving complex (OEC). The OEC consists of a tetranuclear manganese cluster and is the only known biological system capable of oxidizing water (Blakenship 2001).Removal of the manganese cluster allows for study of the mechanism called photoactivation, a light‐and dark‐dependent process by which the OEC assembles. Further study of this process can provide great detail as to repair of PSII following inhibition (G. Dismukes 2008). Removal of the manganese cluster and associated extrinsic proteins also allows unhindered exploration of electron transfer during photochemical events. Plastoquinone A (QA) and plastoquinone B (QB) are electron carriers found in PSII. Despite sharing the same molecular structure, QA and QB exhibit unique redox properties determined by their protein environment. QA participates only in one‐electron transfers, transiently forming paramagnetic semiquinone QA−•(SQA), whereas QB function as a two‐electron gate. Hydrogen bonding from nearby amino acids residues is believed to be partly responsible for such differences in redox properties due to redistribution of electron density. High‐resolution electron paramagnetic resonance (EPR) spectroscopy has the ability to measure magnetic interactions between unpaired electrons and nearby nuclei and provide information about the strength of these interactions (Rutherford, A. W. 1999).Previous pulsed EPR studies established the formation of the H‐bonds between carbonyl oxygens of the QA−• and an amide nitrogen from the protein backbone, and the amino nitrogen, Nd, of an imidazole (Rutherford; Lakshmi). Two‐dimensional pulsed EPR of the SQA in Mn depleted, CN‐treated PSII at pH 9.8, resolved five pairs of cross‐peaks from different types of protons (Lakshmi). They were assigned to the methyl, β‐methylene, and ring protons of the SQA and two protons of the H‐bonds. Unfortunately, these assignments were not verified by the influence of 1H2O/2H2O exchange on the spectra. As a consequence, the assignment of the couplings is not consistent with the values obtained in the DFT calculations (T.‐J. Lin and P. J. O'Malley 2011). In this work we performed 2D pulsed EPR with the SQA in the PS II samples in 1H2O and 2H2O buffered solution in order to unambiguously identify contribution of the exchangeable protons and to determine corresponding hyperfine couplings. These results form a more supported basis for comparison with the results of theoretical computations.Support or Funding InformationPulsed EPR studies were supported by DE‐FG02‐08ER15960 Grant from Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Sciences, US DOE (S.A.D.), and NCRR/NIH Grants S10‐RR15878 and S10‐RR025438 for pulsed EPR instrumentation. Continuous wave EPR studies were supported by NSF‐MRI CHE‐1229498.