Targeted mutagenesis to investigate the role of low-molecular-weight proteins and a bicarbonate cofactor on the assembly and function of Photosystem II

Abstract

Photosystem II (PS II) catalyses the oxidation of water at ambient temperatures and the formation of plastoquinol using light. This multisubunit complex functions as a dimer, each monomer is surrounded by 13 low-molecular-weight (LMW) proteins and includes the Mn4CaO5 oxygen-evolving complex (OEC) and an abundance of other cofactors. The roles of the LMW proteins PsbH, PsbX, and PsbY and their transmembrane interactions with the Sll0933 assembly factor were investigated by the creation and physiological characterisation of strains lacking these proteins in Synechocystis sp. PCC 6803. Removal of the PsbH protein destabilised PS II causing a weakened binding of the chlorophyll a-binding protein CP47 and significant alterations to electron transport reactions at both the acceptor side and donor side of PS II. The PsbH and PsbX proteins appear to have a role in the function of QB (the secondary quinone electron acceptor of PS II) or the QB-binding site. Inactivation of the Sll0933 assembly factor and PsbY failed to produce a strong phenotype but revealed minor effects on assembly and function of PS II. The PsbH, PsbX, and PsbY proteins all show signs of transmembrane interactions with the Sll0933 assembly factor. The PsbH protein was shown to be essential to PS II in the absence of the Sll0933 assembly factor, inactivation of both PsbH and the Sll0933 assembly factor renders Synechocystis sp. PCC 6803 an obligate heterotroph. Recent high-resolution structures of PS II have confirmed bicarbonate is a bidentate ligand to the non-heme iron located between the primary quinone electron acceptor QA and the QB electron acceptor. The absence of PsbH, PsbX, and PsbY all caused a conformational change in the bicarbonate binding environment leading to altered acceptor side electron transport. The D2 protein role in the binding of bicarbonate was investigated due to its interaction with the PsbH and PsbX proteins. Physiological characterisation revealed a conformational change in the bicarbonate binding environment in mutants carrying amino acid substitutions at the D2-Glu242, D2-Lys264, D2-Thr243, and D2-Tyr244 residues suggesting that the precise binding of bicarbonate is required for efficient electron transfer between QA and QB and the associated protonation reactions. However, only mutants carrying substitutions at D2-Thr243 and D2-Tyr244 showed signs of an inhibited formation of plastoquinol due to a disrupted protonation pathway. A considerable effect on PS II assembly was also observed in the D2 mutants.