Time resolved mid-IR spectroscopy facilitates selective detection of transoid-cis and transoid-trans species.

The photophysical properties of betanin in aqueous and alcoholic solutions were determined at room temperature using ultrafast UV-vis-NIR transient absorption spectroscopy (λexc = 535 nm). Its S1 → Sn (n > 1) absorption bands appear with maxima at about λ ∼ 450 and 1220 nm. The short betanin S1 state lifetime (6.4 ps in water) is mainly determined by the efficient S1 → S0 radiationless relaxation, probably requiring a strong change in geometry, since the S1 lifetime grows to 27 ps in the more viscous ethylene glycol. The fluorescence quantum yield is very low (Φf ∼ 0.0007 in water), therefore this deactivation path is of minor importance. Other processes, such as S1 → T1 intersystem crossing or photoproduct formation, are virtually absent, since full S0 ← S1 ground state recovery is observed within tens of picoseconds after photoexcitation. The observed fast light-to-heat conversion in the absence of triplet excited state formation supports the idea that betanin is a photoprotector in vivo.

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 .

Fast radiationless S1 → S0 transition in photo-excited betaxanthins is due to conical intersection seam between S1 and S0 surfaces.