Site-Directed Mutagenesis of Basic Arginine Residues 305 and 342 in the CP 43 Protein of Photosystem II Affects Oxygen-Evolving Activity in Synechocystis 6803

• Published in: Biochemistry (Easton) 1999-02, Vol.38 (5), p.1582-1588
• Main Authors: Knoepfle, Nicholas, Bricker, Terry M, Putnam-Evans, Cindy
• Format: Article
• Language: English
• Subjects: Arginine - genetics; Benzoquinones - chemistry; Chlorophyll - metabolism; Cyanobacteria - genetics; Cyanobacteria - growth & development; Cyanobacteria - metabolism; Electron Transport; Genotype; Mutagenesis, Site-Directed; Oxygen - metabolism; Photosynthetic Reaction Center Complex Proteins - chemistry; Photosynthetic Reaction Center Complex Proteins - genetics; Photosynthetic Reaction Center Complex Proteins - metabolism; Photosystem II Protein Complex; Synechocystis; Water - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9821418

The intrinsic chlorophyll protein CP 43, a component of photosystem II (PS II) in higher plants, green algae, and cyanobacteria, is encoded by the psbC gene. Oligonucleotide-directed mutagenesis was employed to introduce mutations into a segment of psbC that encodes the large extrinsic loop E of CP 43 in the cyanobacterium Synechocystis 6803. Two mutations, R305S and R342S, each produced a strain with impaired photosystem II activity. The R305S mutant strain grew photoautotrophically at rates comparable to the control strain. Immunological analyses of a number of PSII components indicated that this mutant accumulated normal quantities of PSII proteins. However, this mutant evolved oxygen to only 70% of control rates at saturating light intensities. Measurements of total variable fluorescence yield indicated that this mutant assembled approximately 70% of the PSII centers found in the control strain. The R342S mutant failed to grow photoautotrophically and exhibited no capacity for oxygen evolution. However, when grown photoheterotrophically in medium containing both glucose and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), oxygen-evolving activity was observed in the R342S mutant, but at a low level of approximately 10% of the control rate. Immunological analysis of isolated thylakoid membranes from this mutant also indicated that this strain accumulated normal amounts of PSII core proteins. Total variable fluorescence yields for the R342S mutant indicated that it assembled a severely reduced number of fully functional PSII centers. R305S and R342S mutant strains exhibited, respectively, 2.7- and 4-fold increased sensitivity to photoinactivation. The fluorescence rise times for both mutants were comparable to the control when hydroxylamine was used as electron donor. However, both strains exhibited an increase (2.5- and 8-fold, respectively, for R305S and R342S) in fluorescence rise times with water as an electron donor. These results suggest that the mutations R305S and R342S each produce a defect associated with the oxygen-evolving complex of photosystem II. These are the first site-directed mutations in CP 43 to show such an effect.