Photosynthesis requires various photoprotective mechanisms for survival of organisms in high light. In cyanobacteria exposed to high light, the Orange Carotenoid Protein (OCP) is reversibly photoswitched from the orange (OCPO) to the red (OCPR) form, the latter binds to the antenna (phycobilisomes, PBs) and quenches its overexcitation. OCPR accumulation implicates restructuring of a compact dark-adapted OCPO state including detachment of the N-terminal extension (NTE) and separation of protein domains, which is reversed by interaction with the Fluorescence Recovery Protein (FRP). OCP phototransformation supposedly occurs via an intermediate characterized by an OCPR-like absorption spectrum and an OCPO-like protein structure, but the hierarchy of steps remains debatable. Here, we devise and analyze an OCP variant with the NTE trapped on the C-terminal domain (CTD) via an engineered disulfide bridge (OCPCC). NTE trapping preserves OCP photocycling within the compact protein structure but precludes functional interaction with PBs and especially FRP, which is completely restored upon reduction of the disulfide bridge. Non-interacting with the dark-adapted oxidized OCPCC, FRP binds reduced OCPCC nearly as efficiently as OCPO devoid of the NTE, suggesting that the low-affinity FRP binding to OCPO is realized via NTE displacement. The low efficiency of excitation energy transfer in complexes between PBs and oxidized OCPCC indicates that OCPCC binds to PBs in an orientation suboptimal for quenching PBs fluorescence. Our approach supports the presence of the OCPR-like intermediate in the OCP photocycle and shows effective uncoupling of spectral changes from functional OCP photoactivation, enabling redox control of its structural dynamics and function.
Keywords: Fluorescence recovery protein; Orange carotenoid protein; Photoactivation mechanism; Photoprotection; Phycobilisome quenching; Protein-protein interactions.
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