Mass spectrometry-based cross-linking study shows that the Psb28 protein binds to cytochrome b₅₅₉ in Photosystem II

Photosystem II (PSII), a large pigment protein complex, undergoes rapid turnover under natural conditions. During assembly of PSII, oxidative damage to vulnerable assembly intermediate complexes must be prevented. Psb28, the only cytoplasmic extrinsic protein in PSII, protects the RC47 assembly intermediate of PSII and assists its efficient conversion into functional PSII. Its role is particularly important under stress conditions when PSII damage occurs frequently. Psb28 is not found, however, in any PSII crystal structure, and its structural location has remained unknown. In this study, we used chemical cross-linking combined with mass spectrometry to capture the transient interaction of Psb28 with PSII. We detected three cross-links between Psb28 and the α- and β-subunits of cytochrome b ₅₅₉, an essential component of the PSII reaction-center complex. These distance restraints enable us to position Psb28 on the cytosolic surface of PSII directly above cytochrome b₅₅₉, in close proximity to the Q_b site. Protein-protein docking results also support Psb28 binding in this region. Determination of the Psb28 binding site and other biochemical evidence allow us to propose a mechanism by which Psb28 exerts its protective effect on the RC47 intermediate. This study also shows that isotope-encoded cross-linking with the "mass tags" selection criteria allows confident identification ofmore cross-linked peptides in PSII than has been previously reported. This approach thus holds promise to identify other transient protein- protein interactions in membrane protein complexes.