Structural basis of the two-photon photoactivation mechanism of orange carotenoid protein

Cyanobacteria have produced Earth’s oxygen for 2.4 billion years by adapting to fluctuating irradiance. This adaptation relies on orange carotenoid protein (OCP), which mediates light-intensity– dependent photoprotective energy dissipation using a unique two-photon absorption mechanism. Photon absorption by ground-state OCP (OCP ^O ) generates a metastable intermediate (OCP ^1hν ) that either relaxes thermally or, upon absorption of a second photon within ∼1 s, converts to the active photoprotective state (OCP ^R ). By integrating static and time-resolved crystallography, cryo-EM, computation, spectroscopy and biochemistry, we assign the structure of OCP ^1hν , establish its functional relevance and capture structural snapshots along the OCP ^O →OCP ^1hν and OCP ^1hν →OCP ^R photochemical pathways. We elucidate the molecular mechanism of OCP, which serves as a unique biological circuit breaker protecting the photosynthetic machinery from high light flux.