FUNCTIONALLY IMPORTANT DOMAINS OF THE LARGE HYDROPHILIC LOOP OF CP47 AS PROBED BY OLIGONUCLEOTIDE-DIRECTED MUTAGENESIS IN SYNECHOCYSTIS SP PCC-6803

The chlorophyll a-binding protein CP47 serves as core antenna to photosystem II (PS II). The predicted topology of CP47 exhibits six membrane-spanning regions and a large hydrophilic loop (loop E) which rougly includes 200 residues (255-455) and is presumably exposed to the lumenal side of the thylakoid membrane. Several lines of experimental evidence suggest that loop E might be involved in binding or stabilizing functional manganese in the catalytic site of water oxidation or in interacting with the extrinsic PS II-O protein (the 33-kDa manganese-stabilizing protein). To scan loop E for functionally important domains, oligonucleotide-directed mutagenesis has been used to introduce deletions of 3-8 residues in conserved and charged regions of loop E. In addition, one single-site mutation of the only histidine present in loop E was created (H343L). Domains deleted in DELTA1 (1265-F268), DELTA2 (T271-K277), DELTA4 (T304-L309), DELTA5 (F311-N317), and DELTA12 (D440-P447) are required for stable assembly of functional PS II complexes. Deletion of domains DELTA3 (K277-E283) and DELTA11 (R422-E428) significantly reduces the level of assembled PS II and impairs photoautotrophic growth and oxygen evolution. Deletion of domain DELTA8 (A373-D380) enhances the susceptibility to photoinhibition. In contrast, deletion of domains DELTA6 (G333-1336), DELTA7 (K347-R352), DELTA9 (V392-Q394), and DELTA10 (D416-F420) and mutation of H343 to leucine do not seem to severly interrupt PS II structure and function, although all mutants exhibit a slightly decreased stability of PS II as compared to the wild type. Thus, selected domains of the large hydrophilic loop of CP47 are important for PS II structure and function. With respect to possible sites of interaction between loop E of CP47 and the extrinsic PS II-O protein, our results indicate that none of the deletions in the region from residue 330 to 420 (DELTA6, DELTA7, DELTA8, DELTA9, DELTA10) completely interrupts a functional association of the manganese-stabilizing protein to PS II, although the binding characteristics might be changed in some cases.