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 previ ously by biochemical methods that the domain (364)E- D-440 of CP 47 in teracts 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 Syn echocystis 6803, mutations at 17 conserved charged residues were intro duced into the domain (364)E-(444)R Of the CP 47 protein. Only mutatio ns introduced at positions (384)R and (385)R led to a modified PS II p henotype. We previously described a mutation at (RR384385GG) which res ulted in a mutant with a defective oxygen-evolving complex [Putnam-Eva ns, C., & Bricker, T. M. (1992) Biochemistry 31, 11482-11488]. An addi tional 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 th e mutants R384G, R385G, and RR384385EE, respectively. Steady state oxy gen evolution measurements and quantum yield measurements demonstrated that these mutants exhibited significant alterations in their ability to evolve oxygen. Total fluorescence yield measurements indicated tha t all of these mutants contained about 85%-90% of the PS II reaction c enters found in the control strain. This decrease was insufficient to explain the oxygen evolution results. Analysis of oxygen flash yield p arameters indicated that there was little change in the S-state parame ters alpha, beta, gamma, or delta. Measurement of the S-2 lifetime, ho wever, demonstrated that the S-2 lifetime of the mutants was 2-3 times longer than that of the control. Additionally, examination of the ris etime of the oxygen signal indicated that there was a significant reta rdation (6-7-fold) in the rate of oxygen release, suggesting a retarde d S-3-[S-4]-S-0 transition. These data reinforce our hypothesis that t he 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 oxidat ion. We speculate that this positive charge density may be an importan t factor in establishing the proper interaction between CP 47 and the 33 kDa extrinsic protein.