Abstract
Photosystem II (PS II) is a pivotal photochemical oxidoreductase enzyme in photosynthesis, catalysing the oxidation of water and orchestrating electron transfer plastoquinone electron acceptor (QA) and subsequently progressing to the secondary acceptor (QB) ultimately forming plastoquinol (PQH2). This entire process occurs in the presence of light and at ambient temperatures, highlighting the remarkable efficiency of PS II in driving essential reactions within the photosynthetic pathway. PS II, functioning as a dimer, comprises numerous subunits, including 13 low-molecular-weight (LMW) proteins, the Mn4CaO5 oxygen-evolving complex (OEC), and various cofactors. Within PS II, CP43, a chlorophyll-binding protein, and the D2 DE loop emerged as key subunits in facilitating efficient function and assembly of PS II. The PS II reaction centre (RC) comprises the two major proteins, D1 and D2, forming a heterodimer. The DE loop connecting the transmembrane helices D and E of the D2 protein is highly conserved across oxygenic organisms. The focus of this research was to investigate the roles of CP43 and the D2 DE loop in PS II assembly and function. The research aimed to understand how specific conserved residues in CP43 and the D2 DE loop influence PS II assembly, electron transfer, and photoprotection. These conserved residues in CP43 and the D2 DE loop were substituted in Synechocystis sp. PCC 6803 to investigate their roles in PS II assembly and function. Two highly conserved histidine residues involved in chlorophyll binding, CP43-His40 and CP43-His151, situated in close proximity to β-carotenes were selected. Additionally, a bicarbonate molecule, bound to a non-heme iron (NHI) between QA and QB, that has been implicated in a hydrogen bonding network that facilitates electron and proton transfer was studied. The hydrogen bond network is comprised of numerous residues within the D2 DE loop. The residues within the DE loop of the D2 polypeptide, including D2-Met246, D2-Val247, D2-Asn250, and D2-Ser254 were specifically chosen for investigation due to their proximity to QA and PsbT, a LMW protein. Additionally, D2-Arg265, an arginine cap located on the E-helix of D2, was targeted because of its proximity to the non-heme iron (NHI). In CP43 and D2 mutant strains, a consistent observation was impaired PS II assembly/repair and modified acceptor-side electron transfer kinetics. Substitutions of residues near QA influenced donor-side electron transfer processes. Most mutants adapted to high-light conditions in the presence of bicarbonate, but CP43-H40A, CP43-H151Y, D2-M246A, D2-S254H, and D2-V247D remained sensitive. Accumulation of CP43 preassembly modules was a recurrent theme in CP43 and D2 mutants. These results highlight the crucial roles of CP43 in photoprotective mechanisms and the D2 DE loop in stabilising bicarbonate-NHI interactions and QA binding. This research contributes to the broader understanding of PS II assembly and function, suggesting further investigation into these mechanisms could enhance photosynthetic efficiency and resilience in phototrophic organisms.