The orange carotenoid protein (OCP) plays a photoprotective
role in cyanobacterial photosynthesis similar to that of nonphotochemical
quenching in higher plants. Under high-light conditions, the OCP binds to
the phycobilisome (PBS) and reduces the extent of transfer of energy to the
photosystems. The protective cycle starts from a light-induced activation of
the OCP. Detailed information about the molecular mechanism of this
process as well as the subsequent recruitment of the active OCP to the
phycobilisome are not known. We report here our investigation on the OCP
photoactivation from the cyanobacterium Synechocystis sp. PCC 6803 by using a combination of native electrospray mass
spectrometry (MS) and protein cross-linking. We demonstrate that native MS can capture the OCP with its intact pigment and
further reveal that the OCP undergoes a dimer-to-monomer transition upon light illumination. The reversion of the activated
form of the OCP to the inactive, dark form was also observed by using native MS. Furthermore, in vitro reconstitution of the
OCP and PBS allowed us to perform protein chemical cross-linking experiments. Liquid chromatography−MS/MS analysis
identified cross-linking species between the OCP and the PBS core components. Our result indicates that the N-terminal domain
of the OCP is closely involved in the association with a site formed by two allophycocyanin trimers in the basal cylinders of the
phycobilisome core. This report improves our understanding of the activation mechanism of the OCP and the structural binding
site of the OCP during the cyanobacterial nonphotochemical quenching process.