Unifying Perspective of the Ultrafast Photodynamics of Orange Carotenoid Proteins from Synechocystis : Peril of High-Power Excitation, Existence of Different S* States, and Influence of Tagging.

A substantial number of Orange Carotenoid Protein (OCP) studies have aimed to describe the evolution of singlet excited states leading to the formation of a photoactivated form, OCP R . The most recent one suggests that 3 ps-lived excited states are formed after the sub-100 fs decay of the initial S 2 state. The S* state, which has the longest reported lifetime of a few to tens of picoseconds, is considered to be the precursor of the first red photoproduct P 1 . Here, we report the ultrafast photodynamics of the OCP from Synechocystis PCC 6803 carried out using visible-near infrared femtosecond time-resolved absorption spectroscopy as a function of the excitation pulse power and wavelength. We found that a carotenoid radical cation can form even at relatively low excitation power, obscuring the determination of photoactivation yields for P 1 . Moreover, the comparison of green (540 nm) and blue (470 nm) excitations revealed the existence of an hitherto uncharacterized excited state, denoted as S ∼ , living a few tens of picoseconds and formed only upon 470 nm excitation. Because neither the P 1 quantum yield nor the photoactivation speed over hundreds of seconds vary under green and blue continuous irradiation, this S ∼ species is unlikely to be involved in the photoactivation mechanism leading to OCP R . We also addressed the effect of His-tagging at the N- or C-termini on the excited-state photophysical properties. Differences in spectral signatures and lifetimes of the different excited states were observed at a variance with the usual assumption that His-tagging hardly influences protein dynamics and function. Altogether our results advocate for the careful consideration of the excitation power and His-tag position when comparing the photoactivation of different OCP variants and beg to revisit the notion that S* is the precursor of photoactivated OCP R .