title: Identification of functional domains of the extrinsic 12 kDa protein in red algal PSII by limited rroteolysis and directed mutagenesis.
abstract: The extrinsic 12 kDa protein in red algal photosystem II (PSII) functions to minimize the chloride and calcium requirement of oxygen-evolving activity [Enami et al. (1998) Biochemistry 37: 2787]. In order to identify functional domains of the 12 kDa protein, we prepared the 12 kDa protein lacking N-terminal peptides or C-terminal peptides or both by limited proteolysis and directed mutagenesis. The resulting 12 kDa protein fragments were examined for their binding and functional properties by reconstitution experiments. (1) A peptide fragment from Gly-6 to C-terminus of the 12 kDa protein was prepared by V8 protease. This fragment rebound to PSII completely, and it reactivated oxygen evolution partially in the absence of Cl(-) and Ca(2+) ions but significantly in the presence of Cl(-) ion. (2) A peptide from Leu-10 to Phe-83 was obtained by chymotrypsin treatment. This peptide rebound to PSII effectively, but the rebinding did not restore oxygen evolution in both the absence and presence of Cl(-) and Ca(2+) ions. (3) Two mutant proteins, one lacking five residues and the other lacking nine residues of the N-terminus, were able to bind to PSII effectively. Recovery of oxygen evolution by their binding was almost the same as that reconstituted with the V8 protease-treated peptide. (4) Three mutant proteins lacking ten, seven or three residues of the C-terminus effectively rebound to PSII, but their binding did not result in recovery of the oxygen evolution. In contrast, reconstitution with a mutant protein lacking one residue of the C-terminus showed the same high restoration of oxygen evolution as reconstitution with the full-length 12 kDa protein. (5) These results indicate that two residues from lysine of the C-terminus of the 12 kDa protein constitute an important domain for minimizing the chloride and calcium requirement of oxygen evolution. In addition, the N-terminus of the protein, at least five residues, has a secondary function for the chloride requirement.
authors.affiliation: Department of Biology, Faculty of Science, Science University of Tokyo, Kagurazaka 1-3, Shinjuku-ku, Tokyo, 162-8601 Japan.
authors.forename: A
authors.initials: A
authors.lastname: Okumura
authors.forename: H
authors.initials: H
authors.lastname: Ohta
authors.forename: Y
authors.initials: Y
authors.lastname: Inoue
authors.forename: I
authors.initials: I
authors.lastname: Enami
mesh_terms: D020418:Algal Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D002122:Calcium Chloride / Q000494:pharmacology; D000458:Cyanobacteria / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D010101:Oxygen Consumption; D045322:Photosynthetic Reaction Center Complex Proteins / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D011485:Protein Binding; D000461:Rhodophyta / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism
chemical_list: D020418:Algal Proteins; D003574:Cytochrome c Group; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; C064388:oxygen-evolving complex, 33 kDa protein; C031732:cytochrome C-550; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D002122:Calcium Chloride; D010100:Oxygen