Investigating the roles of specific amino acid substitutions found in the D1' and far-red forms of the Photosystem II reaction centre D1 protein

Abstract

Photosystem II is a highly conserved membrane-protein complex that plays an integral role in oxygenic photosynthesis as a light-driven water-plastoquinone oxidoreductase. One of the most important proteins within Photosystem II that contributes to oxygenic photosynthesis is the psbA-encoded D1 protein. The psbA gene encoding D1 is highly conserved in plants and all photosynthetic eukaryotes; however, multiple copies of psbA encoding different forms of the D1 protein are present in cyanobacteria. These copies have been shown to be upregulated under specific conditions, thus allowing them to respond to changing environmental stimuli. Seven distinct isoforms of D1 have been identified by phylogenetic studies. Of these, the current investigation focused on two: the D1' isoform that is expressed under low-oxygen conditions, and the far-red D1 isoform, which is expressed as part of a far-red light acclimation response. Point and combined mutants conveying amino acid changes associated with D1' and far-red D1 were constructed in Synechocystis sp. PCC 6803 and characterised to determine how the structural and physiological function of D1 changed. The D1'-associated point mutants were A81T, N87A, P162S and P173M. Results from these mutants found the introduced amino acid changes impaired electron movement through PS II, decreasing oxygen-evolving activity. Results supported the conclusion that potential advantages for the expression of D1' would be the ability to adapt to a changing aerobic/anaerobic environment or the ability to produce oxygen at the same rate it is consumed in an environment. The far-red D1-associated combined mutants were L114M, L114M:V115I, L114M:V115I:V116G, L114M:V115I:V116G:F119Y and L114M:V115I:V116G:F119Y:L120I. Results showed these mutants all had impaired PS II activity, which resulted in decreased oxygen evolution and ultimately decreased the photoautotrophic growth rates of some strains. The far-red D1-associated point mutants were L120I and I121P, which showed a similar but less pronounced phenotype to the combined mutants. These results supported the conclusion that amino acid substitutions found in the far red form of D1 likely convey a conserved purposed and function. Lastly, a spontaneous double mutant L114M:A251V was characterised. Results showed the L114M mutant had incorporated a deleterious secondary mutation (Ala251 to Val) and potential explanations for why this occurred were explored. The current investigation successfully identified changes to the function of D1 introduced by conserved amino acid substitutions found in the sequence of the D1' and far-red D1 isoforms of D1.