Photoactivation of the orange carotenoid protein requires two light-driven reactions mediated by a metastable...

The orange carotenoid protein (OCP) functions as a sensor of the ambient light intensity and as a quencher of bilin excitons when it binds to the core of the cyanobacterial phycobilisome. We show herein that the photoactivation mechanism that converts the resting, orange-colored state, OCP^O, to the active red-colored state, OCP^R, requires a sequence of two reactions, each requiring absorption of a single photon by an intrinsic ketocarotenoid chromophore. Global analysis of absorption spectra recorded during continuous illumination of OCP^O preparations from Synechocystis sp. PCC 6803 detects the reversible formation of a metastable intermediate, OCP^I, in which the ketocarotenoid canthaxanthin exhibits an absorption spectrum with a partial red shift and a broadened vibronic structure compared to that of the OCP^O state. While the dark recovery from OCP^R to OCP^I is a first-order, unimolecular reaction, the subsequent conversion of OCP^I to the resting OCP^O state is bimolecular, involving association of two OCP^O monomers to form the dark-stable OCP^O dimer aggregate. These results indicate that photodissociation of the OCP^O dimer to form the monomeric OCP^O intermediate is the first step in the photoactivation mechanism. Formation of the OCP^O monomer from the dimer increases the mean value and broadens the distribution of the solvent-accessible surface area of the canthaxanthin chromophore measured in molecular dynamics trajectories at 300 K. The second step in the photoactivation mechanism is initiated by absorption of a second photon, by canthaxanthin in the OCP^O monomer, which obtains the fully red-shifted and broadened absorption spectrum detected in the OCP^R product state owing to displacement of the C-terminal domain and the translocation of canthaxanthin more than 12 Å into the N-terminal domain. Both steps in the photoactivation reaction of OCP are likely to involve changes in the structure of the C-terminal domain elicited by excited-state conformational motions of the ketocarotenoid.