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.
1
A. Wilson, C. Punginelli, A. Gall, C. Bonetti, M. Alexandre, J. M. Routaboul,
C. A. Kerfeld, R. Van Grondelle, B. Robert, J. T. M. Kennis and D. Kirilovsky,
A photoactive carotenoid protein acting as light intensity sensor, Proc.
Natl. Acad. Sci. U. S. A., 2008, 105, 12075–12080.
2
T. Polívka, C. A. Kerfeld, T. Pascher and V. Sundström, Spectroscopic
properties of the carotenoid 3′-hydroxyechinenone in the orange
carotenoid protein from the cyanobacterium Arthrospira maxima, Biochemistry,
2005, 44, 3994–4003.
3
D. Kirilovsky, Photoprotection in cyanobacteria: The orange carotenoid protein
(OCP)-related non-photochemical-quenching mechanism, Photosynth. Res.,
2007, 93, 7–16.
4
V. U. Chukhutsina, J. M. Baxter, A. Fadini, R. M. Morgan, M. A. Pope, K. Maghlaoui,
C. M. Orr, A. Wagner and J. J. van Thor, Light activation of Orange Carotenoid
Protein reveals bicycle-pedal single-bond isomerization, Nat. Commun.,
, DOI:10.1038/s41467-022-34137-4.
5
E. Kish, M. M. M. Pinto, D. Kirilovsky, R. Spezia and B. Robert, Echinenone
vibrational properties: From solvents to the orange carotenoid protein, Biochim.
Biophys. Acta - Bioenerg., 2015, 1847, 1044–1054.
6
I. A. Yaroshevich, E. G. Maksimov, N. N. Sluchanko, D. V. Zlenko, A. V.
Stepanov, E. A. Slutskaya, Y. B. Slonimskiy, V. S. Botnarevskii, A. Remeeva, I.
Gushchin, K. Kovalev, V. I. Gordeliy, I. V. Shelaev, F. E. Gostev, D.
Khakhulin, V. V. Poddubnyy, T. S. Gostev, D. A. Cherepanov, T. Polívka, M.
Kloz, T. Friedrich, V. Z. Paschenko, V. A. Nadtochenko, A. B. Rubin and M. P.
Kirpichnikov, Role of hydrogen bond alternation and charge transfer states in
photoactivation of the Orange Carotenoid Protein, Commun. Biol.,
2021, 4, 1–13.
7 V.
Šlouf, V. Kuznetsova, M. Fuciman, C. B. de Carbon, A. Wilson, D. Kirilovsky and
T. Polívka, Ultrafast spectroscopy tracks carotenoid configurations in the
orange and red carotenoid proteins from cyanobacteria, Photosynth. Res.,
2017, 131, 105–117.