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 364E-440D 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 364E-444R of the CP 47 protein. Only mutations introduced at positions 384R and 385R 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, 384R to 384G, 385R to 385G, and 384,385RR to 384,385EE 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 S2 lifetime, however, demonstrated that the S2 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 S3-[S4]-S0 transition. These data reinforce our hypothesis that the positive charge density at positions 384R and 385R 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 33kDa extrinsic protein.

The intrinsic chlorophyll-protein CP 47 is a component of photosystem II which functions in both light-harvesting and oxygen evolution. The large extrinsic loop E of this protein has been shown to interact with the oxygen-evolving site. Previously, Vermaas and coworkers have produced a number of deletions within loop E which yielded mutants which were unable to grow photoautotrophically and which could not evolve oxygen at normal rates. During the course of our site-directed mutagenesis program in Synechocystis 6803, we have altered all of the conserved charged residues which were present within six of these deletions. All ten of these mutants were photoautotrophic and evolved oxygen at normal rates. We speculate that the severe phenotypes of the deletion mutants observed by Vermaas and coworkers is due to large structural perturbations in the extrinsic loop E of CP 47.

The ubiquinol-cytochrome c2 oxidoreductases (cytochrome bc1 complex) of Rhodobacter sphaeroides contains highly conserved cytochrome b, cytochrome c1 and Rieske FeS subunits, as well as a unique 14 kDa polypeptide, designated as subunit IV, thought to function as a ubiquinol-binding protein [Yu and Yu (1991) Biochemistry 30, 4934-4939]. As the topology of subunit IV is unknown and that of the FeS subunit remains a matter of debate, both the inner (cytoplasmic) and outer (periplasmic) surfaces of the intracytoplasmic membrane (ICM) were digested with proteinase K, and cleavage products were identified by immunoblotting. In uniformly oriented chromatophore vesicles (inner ICM surface exposed), fragments of approx. 4 and 1 kDa were removed from subunit IV and the FeS protein respectively. Neither subunit IV nor the FeS protein was cleaved from the outer ICM surface as exposed in osmotically protected spheroplasts or as presented to proteinase K after microencapsulation of the protease in unilamellar liposomes and fusion of these structures to chromatophore vesicles. Studies with the isolated bc1 complex, however, suggested that the C-terminal domain of the Rieske FeS, thought to reside on the periplasmic side of the ICM, was resistant to proteinase K. Overall, these results suggest a single N-terminal transmembrane helix for the FeS protein, with exposure of the N-terminus to the cytoplasm and an orientation in which a major, N-terminal portion of subunit IV is located in the cytoplasm with the predicted C-terminal transmembrane domain anchoring this polypeptide to the membrane.