Characterization of the light-induced cross-linking of the alpha-subunit of cytochrome b559 and the D1 protein in isolated photosystem II reaction centers.
Illumination of the isolated reaction center of photosystem II generates a protein of 41 kDa molecular mass. Using immunoblotting, it is confirmed that the protein is an adduct of the D1 protein and the alpha-subunit of cytochrome b559. Its formation seems to be photochemically induced, being independent of temperature between 4 and 20 degrees C and unaffected by a mixture of protease inhibitors. The maximum levels are detected when the pH is in the region 6.5-8.5 and when illumination intensities are moderate. Although higher light intensities induce a higher rate of formation, the accumulation of elevated levels of the 41-kDa protein does not occur due to light-induced degradation. This degradation is also unaffected by the presence of protease inhibitors. Proteolytic mapping and N-terminal sequencing indicates that the cross-linking process involves the N-terminal serine of the alpha-subunit of cytochrome b559 and D1 residues in the 239-244 FGQEEE motif close to the QB binding site. In conclusion, the results indicate that the N terminus of the alpha-subunit is exposed on the stromal side of photosystem II in such a way as to undergo light-induced cross-linking in the QB region of the D1 protein. They also suggest that the 41-kDa adduct may be an intermediate before the light-induced cleavage of the D1 protein in the FGQEEE region.
Barbato
R
R
Biochemistry Department, Wolfson Laboratories, Imperial College of Science, Technology & Medicine, London, United Kingdom.
Friso
G
G
Ponticos
M
M
Barber
J
J
D000595:Amino Acid Sequence; D000906:Antibodies; D001665:Binding Sites; D002736:Chloroplasts / Q000378:metabolism*; D003432:Cross-Linking Reagents; D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008027:Light; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D018532:Pisum sativum; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D045332:Photosystem II Protein Complex; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D014908:Triticum
D000906:Antibodies; D003432:Cross-Linking Reagents; D003573:Cytochrome b Group; D046911:Macromolecular Substances; D010446:Peptide Fragments; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; C019313:cytochrome b559