Site-directed mutagenesis of the CP 47 protein of photosystem II: Alteration of conserved charged residues in the domain (364)E-(444)R

The intrinsic chlorophyll-protein CP 47 is a component of photosystem II in higher plants, green algae and cyanobacteria. We had shown previously by biochemical methods that the domain (364)E- D-440 of CP 47 interacts with the 33 kDa extrinsic protein of photosystem II [Odom, W. R., & Bricker, T. M. (1992) Biochemistry 31, 5616-5620]. In this study, using oligonucleotide-directed mutagenesis in the cyanobacterium Synechocystis 6803, mutations at 17 conserved charged residues were introduced into the domain (364)E-(444)R Of the CP 47 protein. Only mutations introduced at positions (384)R and (385)R led to a modified PS II phenotype. We previously described a mutation at (RR384385GG) which resulted in a mutant with a defective oxygen-evolving complex [Putnam-Evans, C., & Bricker, T. M. (1992) Biochemistry 31, 11482-11488]. An additional set of mutations, (384)R to (384)G, (385)R to (385)G, and (384,385)RR to (384,385)EE has now been introduced at this site yielding the mutants R384G, R385G, and RR384385EE, respectively. Steady state oxygen evolution measurements and quantum yield measurements demonstrated that these mutants exhibited significant alterations in their ability to evolve oxygen. Total fluorescence yield measurements indicated that all of these mutants contained about 85%-90% of the PS II reaction centers found in the control strain. This decrease was insufficient to explain the oxygen evolution results. Analysis of oxygen flash yield parameters indicated that there was little change in the S-state parameters alpha, beta, gamma, or delta. Measurement of the S-2 lifetime, however, demonstrated that the S-2 lifetime of the mutants was 2-3 times longer than that of the control. Additionally, examination of the risetime of the oxygen signal indicated that there was a significant retardation (6-7-fold) in the rate of oxygen release, suggesting a retarded S-3-[S-4]-S-0 transition. These data reinforce our hypothesis that the positive charge density at positions (384)R and (385)R in the large extrinsic loop of CP 47 is necessary for its function in water oxidation. We speculate that this positive charge density may be an important factor in establishing the proper interaction between CP 47 and the 33 kDa extrinsic protein.