A Conserved Mechanism for Positioning Ferredoxin NADP+ Reductase at Photosystem I in Green Algae

The association of ferredoxin-NADP+ reductase (FNR) with thylakoid membranes constitutes a central regulatory node in photosynthetic electron transport, governing NADPH production essential for carbon fixation. In cyanobacteria and higher plants, this interaction is mediated by an intrinsic FNR domain or specialized proteins, yet the mode of recruitment in green algae has remained enigmatic. Here, we show that in the green microalga Chlamydomonas reinhardtii, FNR is directly tethered to photosystem I-LHCI (PSI-LHCI) through a conserved N-terminal -helix of the antenna protein Lhca4. Cryogenic electron microscopy localizes FNR to the stromal side of PSI proximal to Lhca4, while AlphaFold modeling identifies a specific interaction interface, which we validate using isothermal titration calorimetry. Structural modeling further reveals that the spatial separation between PSI-bound FNR and ferredoxin (Fd) is incompatible with direct electron transfer, indicating that it occurs sequentially rather than through a stable PSI-Fd-FNR complex. Comparative analysis demonstrates that the FNR-binding N-terminal motif of Lhca proteins is conserved across diverse green microalgae, suggesting an evolutionarily conserved strategy for positioning FNR at PSI. Collectively, our results uncover a novel mechanism for FNR recruitment and establish a new principle by which photosynthetic electron partitioning is regulated through spatial organization of electron transfer components.