Site-directed mutagenesis of glutamate residues in the large extrinsic loop of the photosystem II protein CP 43 affects oxygen-evolving activity and PSII assembly

The psbC gene encodes the intrinsic chlorophyll protein CP 43, a component of photosystem II in higher plants, green algae, and cyanobacteria. Oligonu cleotide-directed mutagenesis was used to introduce mutations into the port ion of psbC that encodes the large extrinsic loop E of CP 43 in the cyanoba cterium Synechocystis 6803. Three mutations, E293Q, E339Q, and E352Q, each produced a strain with impaired photosystem II activity. The E293Q mutant s train grew photoautotrophically at rates comparable to the control strain. Immunological analyses of several PS II components indicated that this muta nt accumulated normal quantities of PS II proteins. However, this mutant ev olved oxygen to only 56% of control rates at saturating light intensities. Measurements of total variable fluorescence yield indicated that this mutan t assembled approximately 60% of the fully functional PS II centers found i n the control strain. The E339Q mutant grew photoautotrophically at a sever ely reduced rate. Both immunological analysis and variable fluorescence yie ld experiments indicated that E339Q assembled a normal complement of PS IT centers. However, this mutant was capable of evolving oxygen to only 20% of control rates. Variable fluorescence yield experiments demonstrated that t his mutant was inefficient at using water as an electron donor. Both E293Q and E339Q strains exhibited an increased (approximately 2-fold) sensitivity to photoinactivation. The E352Q mutant was the most severely affected. Thi s mutant failed to grow photoautotrophically and exhibited essentially no c apacity for oxygen evolution. Measurements of total variable fluorescence y ield indicated that this mutant assembled no functional PS II centers. Immu nological analysis of isolated thylakoid membranes from E352Q revealed a co mplete absence of CP 43 and reduced levels of both the D1 and manganese-sta bilizing proteins. These results suggest that the mutations E293Q and E339Q each produce a defect associated with the oxygen-evolving complex of photo system II. The E352Q mutation appears to affect the stability of the PS II complex. This is the first report showing that alteration of negatively cha rged residues in the CP 43 large extrinsic loop results in mutations affect ing PS II assembly/function.