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The orange carotenoid protein (OCP) serves as a sensor of light intensity and an effector of phycobilisome (PB)–associated photoprotection in cyanobacteria. Structurally, the OCP is composed of two distinct domains spanned by a single carotenoid chromophore. Functionally, in response to high light, the OCP converts from a dark-stable orange form, OCPO, to an active red form, OCPR. The C-terminal domain of the OCP has been implicated in the dynamic response to light intensity and plays a role in switching off the OCP's photoprotective response through its interaction with the fluorescence recovery protein. The function of the N-terminal domain, which is uniquely found in cyanobacteria, is unclear. To investigate its function, we isolated the N-terminal domain in vitro using limited proteolysis of native OCP. The N-terminal domain retains the carotenoid chromophore; this red carotenoid protein (RCP) has constitutive PB fluorescence quenching activity comparable in magnitude to that of active, full-length OCPR. A comparison of the spectroscopic properties of the RCP with OCPR indicates that critical protein–chromophore interactions within the C-terminal domain are weakened in the OCPR form. These results suggest that the C-terminal domain dynamically regulates the photoprotective activity of an otherwise constitutively active carotenoid binding N-terminal domain.

Most cyanobacteria, under high light conditions, decrease the amount of energy arriving at the reaction centers by increasing thermal energy dissipation at the level of the phycobilisome, the extramembranous antenna. This mechanism is induced by photoactivation of the Orange Carotenoid Protein (OCP). To identify how the activated OCP interacts with phycobilisomes (PBs), several OCP mutants were constructed, and the influence of mutations on photoactivity, stability, and binding to PBs was characterized. The disruption of the salt bridge between Arg155 and Glu244, which stabilizes the interaction between the N- and C-terminal domains, increased the rate of photoactivity and the stability of the photoactivated OCP, suggesting that the activated OCP has an open structure with decreased interdomain interaction. Changing Glu244 to leucine had no effect on OCP binding to PBs. By contrast, substitution of Arg155 with a neutral or a negatively charged amino acid largely decreased OCP binding to the PBs, whereas substitution with a lysine slightly perturbed the interaction. These results strongly suggest that the surface of the N-terminal domain, containing the Arg155, interacts with the PB and that the positive charge of Arg155 plays a key role in photoprotection.