The orange carotenoid protein (OCP) is the water-soluble mediator of non-photochemical quenching in cyanobacteria, a crucial photoprotective mechanism in response to excess illumination. OCP converts from a globular, inactive state (OCPo) to an extended, active conformation (OCPr) under high-light conditions, resulting in a concomitant redshift in the absorption of the bound carotenoid. Here, OCP was trapped in either the active or inactive state by fixing each protein conformation in trehalose-sucrose glass. Glass-encapsulated OCPo did not convert under intense illumination and OCPr did not convert in darkness, allowing the optical properties of each conformation to be determined at room temperature. We measured pump wavelength-dependent transient absorption of OCPo in glass films and found that initial OCP photoproducts are still formed, despite the glass preventing completion of the photocycle. By comparison to the pump wavelength dependence of the OCPo to OCPr photoconversion yield in buffer, we show that the long-lived carotenoid singlet-like feature (S*) is associated with ground-state heterogeneity within OCPo, rather than triggering OCP photoconversion.
