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

[Image: see text] 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).