Rubredoxin 1 promotes the proper folding of D1 and is not required for heme b559 assembly in Chlamydomonas II

Photosystem II (PSII), the water:plastoquinone oxidoreduc-tase of oxygenic photosynthesis, contains a heme b559 iron whose axial ligands are provided by histidine residues from the alpha (PsbE) and beta (PsbF) subunits. PSII assembly depends on accessory proteins that facilitate the step-wise association of its protein and pigment components into a functional complex, a process that is challenging to study due to the low accumula-tion of assembly intermediates. Here, we examined the putative role of the iron[1Fe-0S]-containing protein rubredoxin 1 (RBD1) as an assembly factor for cytochrome b559, using the RBD1-lacking 2pac mutant from Chlamydomonas reinhardtii, in which the accumulation of PSII was rescued by the inacti-vation of the thylakoid membrane FtsH protease. To this end, we constructed the double mutant 2pac ftsh1-1, which harbored PSII dimers that sustained its photoautotrophic growth. We purified PSII from the 2pac ftsh1-1 background and found that alpha and beta cytochrome b559 subunits are still present and coordinate heme b559 as in the WT. Interestingly, immunoblot analysis of dark-and low light-grown 2pac ftsh1-1 showed the accumulation of a 23-kDa fragment of the D1 protein, a marker typically associated with structural changes resulting from photodamage of PSII. Its cleavage occurs in the vicinity of a nonheme iron which binds to PSII on its electron acceptor side. Altogether, our findings demonstrate that RBD1 is not required for heme b559 assembly and point to a role for RBD1 in promoting the proper folding of D1, possibly via de-livery or reduction of the nonheme iron during PSII assembly.