Site-directed mutagenesis of glutamate residues in the large extrinsic loop of the photosystem II protein CP 43 affects oxygen-evolving activity and PS II assembly
Abstract
The psbC gene encodes the intrinsic chlorophyll protein CP 43, a component of photosystem II in higher plants, green algae, and cyanobacteria. Oligonucleotide-directed mutagenesis was used to introduce mutations into the portion of psbC that encodes the large extrinsic loop E of CP 43 in the cyanobacterium Synechocystis 6803. Three mutations, E293Q, E339Q, and E352Q, each produced a strain with impaired photosystem II activity. The E293Q mutant strain grew photoautotrophically at rates comparable to the control strain. Immunological analyses of several PS II components indicated that this mutant accumulated normal quantities of PS II proteins. However, this mutant evolved oxygen to only 56% of control rates at saturating light intensities. Measurements of total variable fluorescence yield indicated that this mutant assembled approximately 60% of the fully functional PS II centers found in the control strain. The E339Q mutant grew photoautotrophically at a severely reduced rate. Both immunological analysis and variable fluorescence yield experiments indicated that E339Q assembled a normal complement of PS II centers. However, this mutant was capable of evolving oxygen to only 20% of control rates. Variable fluorescence yield experiments demonstrated that this mutant was inefficient at using water as an electron donor. Both 0 and E339Q strains exhibited an increased (approximately 2-fold) sensitivity to photoinactivation. The E352Q mutant was the most severely affected. This mutant failed to grow photoautotrophically and exhibited essentially no capacity for oxygen evolution. Measurements of total variable fluorescence yield indicated that this mutant assembled no functional PS II centers. Immunological analysis of isolated thylakoid membranes from E352Q revealed a complete absence of CP 43 and reduced levels of both the D1 and manganese- stabilizing proteins. These results suggest that the mutations E293Q and E339Q each produce a defect associated with the oxygen-evolving complex of photosystem II. The E352Q mutation appears to affect the stability of the PS II complex. This is the first report showing that alteration of negatively charged residues in the CP 43 large extrinsic loop results in mutations affecting PS II assembly/function.