Two-Photon-Driven Photoprotection Mechanism in Echinenone-Functionalized Orange Carotenoid Protein

Orange carotenoid protein (OCP) is a photoactive protein that mediates photoprotection in cyanobacteria. OCP binds different ketocarotenoid chromophores such as echinenone (ECN), 3'- hydroxyechinenone (hECN), and canthaxanthin (CAN). In the dark, OCP is in an inactive orange form known as OCP^O; upon illumination, a red active state is formed, referred to as OCP^R, that can interact with the phycobilisome. Large gaps still exist in the mechanistic understanding of the events between photon absorption and formation of the OCP^R state. Recent studies suggested that more than one photon may be absorbed during the photocycle. Using a two-pulse excitation setup with variable time delays between the pulses, we demonstrate that canthaxanthin-functionalized OCP^O forms the OCP^R signature after absorption of a single photon. By contrast, OCP^O complexed with hECN or ECN does not photoconvert to OCP^R upon single photon absorption. Instead, OCP^R is formed only upon absorption of a second photon arriving roughly one second after the first one, implying the existence of a metastable light-sensitive OCP^1hv intermediate. To the best of our knowledge, a sequential 2-photon absorption mechanism in a single biological photoreceptor chromophore is unique. It results in a nonlinear response function with respect to light intensity, effectively generating a threshold switch. In the case of OCP, this prevents down regulation of photosynthesis at low light irradiance.