Abstract
Oxygenic photosynthesis exploits light, water and carbon dioxide to transform light energy into chemical energy. This process is fundamental in shaping the current oxygenic atmosphere and supports most life on earth. The light-dependent reaction of oxygenic photosynthesis uses light energy and converts it into ATP and NADPH, this process involves four major membrane-bound protein complexes; these include Photosystem II, Photosystem I, cytochrome b6f and ATP synthase. Photosystem II is a multi-protein subunit embedded into the thylakoid membrane of plants, algae and cyanobacteria; it is a highly conserved structure with four core protein complexes, including two homologous proteins D1 and D2, and chlorophyll- binding proteins 43 and 47. The D1 protein is encoded by a psbA gene and is highly susceptible to light damage. Cyanobacteria can have multiple copies of this gene and phylogenetic analysis has shown several variants of the D1 protein. The different D1 isoforms can be divided into different groups; however, distribution is not linked to any distinct traits—the cyanobacterium Synechocystis sp. PCC 6803 carries three copies of the psbA gene; psbA2 and psbA3 encode one type of D1, expressed under normal growth and most stress conditions. The psbA1 gene encodes the D1' protein, an early form of D1 expressed under micro-aerobic conditions. Conserved amino acid substitutions in the D1' protein could affect Photosystem II function, altering photosynthesis. The aim of this study was to investigate if the conserved amino acid changes in the low-oxygen expressed D1 protein altered the function and activity of Photosystem II reaction centres, affecting photosynthetic performance. Eight amino acid residues were characterised by introducing these different residues into D1 protein in Synechocystis sp. PCC 6803: these included Gly80, Phe158, Phe186, Ile192, Thr286, Thr292, Met293 and Ala336. Most of the amino acid residues are located either between or in proximity to the chlorophyll molecule P680 and electron carrier Yz, or close to the oxygen-evolving complex of Photosystem II. Overall, 16 mutant strains were generated by Quickchange II
II
polymerase chain reactions; these included a combination of single, double and triple strains. The mutant strains were analysed by measuring the photoautotrophic growth rate, Photosystem II activity, assembly, and electron transfer activity. All strains grew photoautotrophically; however, the growth rate was impaired in the F158L, F186L, T286A and A336V mutant strains. Photosystem II activity in more than half of the mutant strains were altered; F158L, F186L and G80A:F158L strains displayed the most significant reductions. The levels of assembled active Photosystem II reaction centres reduced in all the mutant strains except G80A, I192V and A203T:A336V. The electron transfer activity between QA and QB in most mutant strains was impaired, and back reaction by charge recombination was altered; all mutant strains displayed a donor side effect in Photosystem II, particularly strains with mutations at Gly80, Phe158, Phe186 and Thr286. The results presented demonstrate the importance of the studied residues in Photosystem II’s function, supporting the view that these conserved amino acid residues play a crucial role, either directly or indirectly, in electron transfer activity within the Photosystem II reaction centres. The accumulation of the various amino acid substitutions could provide a D1' that is functionally distinct from normally expressed D1 and confer a selective advantage under specific environmental conditions.