The structure and operation of the binding site for the terminal quinone electron acceptor of Photosystem II

Photosystem II (PS II) is the light-driven oxidoreductase enzyme that performs the essential water-splitting activity of oxygenic photosynthesis. Electrons derived from water oxidation by the photosystem are transferred to the primary (Q_A) and terminal (Q_B) plastoquinone electron acceptors. Export of the electrons derived from water splitting into the electron transport chain involves the transient binding and proton-coupled reduction of Q_B Q_BH₂, necessitating a binding site that can support the necessary reduction reactions and quinone/quinol exchange mechanism. The D1 reaction centre protein of PS II contributes amino acid residues to the Q_B-binding site that include a hydrogen-bonding network with the headgroup of the quinone electron acceptor. Residues connecting the D and E transmembrane helices also contribute to the structure and the function of the binding cavity. However, in vivo study of Q_B-binding to date has not revealed the full contribution of the residues implicated in protonation and exchange by structural studies and QM/MM calculations. The roles of D1 residues in the operation of the Q_B-binding site of PS II were therefore elucidated with a targeted mutagenesis approach in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). D1:Phe265 was revealed to directly tune the Q_B/Q_B^- redox couple via a hydrogen-bonding interaction using its mainchain atoms. Additionally, elevated PS II fluorescence yield was associated with a shift in the reduction of PS I fluorescence in mutants targeting Phe265. Alanine-scanning mutagenesis of a 3₁₀-helix region of the D1 D-E Loop indicated the PG lipid-interfacing residue D1:Phe260 modified Q_A^- to Q_B electron transfer and exhibited a notable recovery phenotype in response to high-light-induced photoinhibition. Mutations targeting the D1:Leu271 residue of the D1 E helix revealed impaired PS II activity associated with a reduced free-energy gap between the Q_A/Q_A^- and Q_B/Q_B^- redox couples, and a re-emergence of the elevated PS II fluorescence yield observed in mutant strains targeting Phe265. Deletion of PS I in the A263R, F265K, and L271A mutants resulted in the attenuation of the observed elevated PS II fluorescence yields to control levels, providing evidence for inter-photosystem energy transfer being the cause of heightened PS II fluorescence in strains with mutations targeting the Q_B-binding site. This informed the proposal of an energy transfer model whereby energy transfer from PS I into PS II, assisted by the phycobilisome, is employed as a response to a dysfunctional Q_B-binding site. Given the high conservation of residues at the Q_B-binding site between Synechocystis and other photosynthetic organisms of both prokaryota and eukaryota, these findings present important implications for understanding and engineering of these organisms upon which the human population relies.