Structural proteomics reveals the functional docking interface of ferredoxin‐ NADP + reductase on photosystem I in the red alga Cyanidioschyzon merolae

SUMMARY Efficient photosynthetic electron transfer relies on transient interactions between photosystem I (PSI) and soluble electron carriers. However, structural information describing these transient interactions are limited in red algae. Here, we applied chemical cross‐linking mass spectrometry (XL‐MS) to isolated thylakoid membranes of the red alga Cyanidioschyzon merolae and generated an interactome map of photosynthetic protein complexes. Using three independent cross‐link identification algorithms, we obtained a high‐confidence dataset of intra/intercomplex interactions. Among them, we identified a cross‐link between K94 of FNR and K108 of the stromal subunit PsaD. Cross‐linking restraint‐guided protein–protein docking using HADDOCK2.4 revealed a direct docking interface for FNR on PsaD side. Structural analysis of the resulting complex indicated that the binding of FNR to PSI is predominantly electrostatic, in which the K4 residue of FNR is involved in making a salt bridge with E91 of PsaD as well as a conventional hydrogen bond with G90 of PsaD. Site‐directed mutagenesis of the FNR K4 residue significantly impaired the NADP + reduction kinetics, as compared with the WT FNR, supporting that FNR binding to PSI is required for efficient FNR and ferredoxin (FDX)‐dependent NADP + photoreduction. These results strongly suggest that FNR binds to the stromal side of PSI in an orientation that enables efficient electron transfer from FDX. Additionally, we detected a cross‐link between the phycobilisome core protein ApcA and PsaD suggesting that it may serve as a shared interaction hub for both FNR and PBS, implying that these interactions could either occur simultaneously or compete for binding while still allowing electron transfer via soluble FDX.