New Insights into Plastocyanin– Cytochrome b ₆ f Formation: the Role of Plastocyanin Phosphorylation

In this work we investigated the role of plastocyanin (PC) phosphorylation in photosynthetic electron transfer, focusing on interactions with both cytochrome- b ₆ f (Cyt b ₆ f ) and Photosystem-I (PSI) in Chlamydomonas reinhardtii . While the binding and electron transfer between PC and PSI are well characterized, the interaction between PC and Cyt f remains less clear. Using chemical cross-linking combined with mass spectrometry, we identified two potential binding models for PC and Cyt f : “Side-on” and “Head-on”. To evaluate electron transfer, we developed an in vitro system that allowed oxidized PC, formed via light-driven electron transfer at PSI, to re-oxidize Cyt f . Our data shows that a phosphomimetic variant of PC, where phosphorylated PC S49 residue interacts with PetA-K188, displays faster Cyt f oxidation, likely optimizing binding and electron transfer between PC and Cyt f . Additionally, PC phosphomimetic variants exhibited slower transfer rates than wild type, suggesting that phosphorylation modulates PC’s interaction with PSI. This regulation likely optimizes Cyt f oxidation and electron transfer under conditions of low PC availability, such as during high light stress. Overall, PC phosphorylation appears to play a role in fine-tuning electron transfer between PSI, Cyt b ₆ f , and PC, thereby ensuring efficient photosynthesis in dynamic environmental conditions.