Characterization of the light-induced cross-linking of the α-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 α-subunit of cytochrome b₅₅₉. Its formation seems to be photochemically induced, being independent of temperature between 4 and 20°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 α-subunit of cytochrome b₅₅₉ and D1 residues in the 239-244 FGQEEE motif close to the Q_B binding site. In conclusion, the results indicate that the N terminus of the α-subunit is exposed on the stromal side of photosystem II in such a way as to undergo light-induced cross-linking in the Q_B 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.