Lateral organization of cytochrome b ₆ f in thylakoid membranes controls photosynthetic electron transfer efficiency

Photosynthetic electron transfer relies on the coordinated function and spatial organization of large protein complexes within the thylakoid membrane. The cytochrome b ₆ f complex ( b ₆ f ) functionally interconnects photosystem (PS) II and PSI in photosynthetic electron transfer and is equally distributed between appressed and non-appressed thylakoid membranes. Here, we investigate the functional link between the lateral distribution of b ₆ f and efficient photosynthetic electron flow in Chlamydomonas reinhardtii . We engineered strains with stromal fusions between PetA of b ₆ f and two fluorescent proteins (FPs) of different molecular mass: Clover and ATeam. Under oxic conditions, these strains exhibited significantly slower electron transfer rates (ETR), lower PSII quantum yields, and increased donor-side limitation of PSI. State transitions were diminished in the fusion strains, accompanied by a strong impairment of STT7-dependent function, suggesting that the presence of fused FPs at b ₆ f interfere with STT7 function. Yet, ETR phenotypes were STT7-independent and FP fusion did not impact intrinsic b ₆ f function. In situ cryogenic electron tomography revealed a significant depletion of b ₆ f from appressed thylakoid membranes in the ATeam strains, while the overall membrane protein concentration remained unchanged. Overall, our data indicate that a balanced distribution of b ₆ f between appressed and non-appressed thylakoid membranes is essential for regulating photosynthetic electron transfer, highlighting the functional importance of thylakoid molecular architecture in vivo.