The Role of Carotenoid Excited State in Orange Carotenoid Protein Activation

Petra Chrupková1,2, Ivo van Stokkum3, Thomas Friedrich4, Marcus Moldenhauer4, Nediljko Budisa5, Hsueh-Wei Tseng5, Tomáš Polívka2, Dmitry A. Cherepanov6,7, Eugene G. Maksimov7, Miroslav Kloz1

1 ELI-Beamlines, Institute of Physics, Na Slovance 2, 182 21 Praha 8, Czech Republic

2University of South Bohemia in České Budějovice, Faculty of Science, Branišovská 1645/31a, 370 05 České Budějovice

3Vrije Universiteit, Department of Physics and Astronomy, Faculty of Sciences, De Boelelaan 1081, 1081HV Amsterdam, The Netherlands

4Technische Universität Berlin, Institute of Chemistry PC 14, Straße des 17. Juni 135, 10623 Berlin, Germany

5University of Manitoba, Department of Chemistry, 144 Dysart Rd, 360 Parker Building, Winnipeg, MB R3T 2N2, Canada

6N.N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences, 142432 Moscow, Russian Federation

7Lomonosov Moscow State University, A.N. Belozersky Institute of Physical-Chemical Biology, 119991 Moscow, Russian Federation

8Lomonosov Moscow State University, Faculty of Biology, Vorobyovy Gory 1-12, Moscow 119991, Russian Federation

The Orange Carotenoid Protein (OCP) is a unique type of protein naturally present in cyanobacteria, serving a photoprotection function. Normally, at daily sunlight levels, the protein remains in its inactive, so-called orange form. However, under extensive photon flux, the protein activates into its "red" form, enabling it to non-photochemically quench excessive energy flowing from the phycobilisome to the reaction center. The photoactivation process exhibits a very low quantum yield (1%), with activation finely tuned for adaptation to fluctuating light levels. This ensures the protein remains inactive under ideal light conditions while effectively managing excessive energy in light-harvesting complexes1–3.

Another aspect that distinguishes the OCP is the inclusion of carotenoids, such as echinenone, hydroxyechinenone, and canthaxanthin, within its core, where they play a crucial role in light absorption and subsequent protein activation. The carotenoids' strong absorbance of blue-green light is exploited as a "sensor" for light conditions. The absorbed energy can be converted into a form of mechanical motion as needed, activating the protein.

There is an ongoing debate regarding the sequence of events following light absorption that leads to the formation of the red form of OCP, with a consensus that the excited state manifold of the carotenoid plays a vital, initiating role4–7. This study aims to elucidate the differences in energy flux between specific excited states of the carotenoid echinenone in various solvents (methanol, acetonitrile, cyclohexane) and when incorporated into the OCP protein.

The investigation utilized Femtosecond Stimulated Raman Spectroscopy (FSRS) in both upshifted and downshifted regions, along with Transient Absorption Spectroscopy. This approach offered a higher level of correlation between vibrational and absorption spectroscopy, enhancing the understanding of the excited state dynamics.

Our findings reveal unique vibrational characteristics of echinenone associated with OCP's photoactivation stages (S2 state). We also identified a notable absence of vibrational signature for echinenone's relaxed S1 state within OCP and observed stronger signals from a highly excited ground state (GS) in OCP. Additionally, the presence of a short-lived intramolecular charge transfer state (ICT) was detected. 

These observations are attributed to the altered conformation of carotenoid once embedded in the protein environment.

The study also puts forward a hypothesis regarding the photoactivation mechanism of the Orange Carotenoid Protein (OCP), highlighting the significant role of an extraordinarily high level of excitation in longitudinal stretching modes as the primary driving force. This suggests that the specific vibrational energy states of carotenoids, influenced by their interaction with the protein environment, are crucial for initiating the photoactivation process that leads to the protective red form of OCP.

 

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