Here, we propose a possible photoactivation mechanism of a 35-kDa blue light-triggered photoreceptor, the Orange Carotenoid Protein (OCP), suggesting that the reaction involves the transient formation of a protonated ketocarotenoid (oxocarbenium cation) state. Taking advantage of engineering an OCP variant carrying the Y201W mutation, which shows superior spectroscopic and structural properties, it is shown that the presence of Trp201 augments the impact of one critical H-bond between the ketocarotenoid and the protein. This confers an unprecedented homogeneity of the dark-adapted OCP state and substantially increases the yield of the excited photoproduct S*, which is important for the productive photocycle to proceed. A 1.37 Å crystal structure of OCP Y201W combined with femtosecond time-resolved absorption spectroscopy, kinetic analysis, and deconvolution of the spectral intermediates, as well as extensive quantum chemical calculations incorporating the effect of the local electric field, highlighted the role of charge-transfer states during OCP photoconversion.

A N Bach Institute of Biochemistry Federal Research Center of Biotechnology of the Russian Academy of Sciences Moscow Russia

A N Belozersky Institute of Physical Chemical Biology Moscow State University Moscow Russia

Chemistry department M 5 Lomonosov Moscow State University Moscow Russia

ELI Beamlines Institute of Physics Praha Czech Republic

European XFEL GmbH Schenefeld Germany

Faculty of Biology M 5 Lomonosov Moscow State University Moscow Russia

Institut de Biologie Structurale J P Ebel Université Grenoble Alpes CEA CNRS Grenoble France

Institute of Biological Information Processing Forschungszentrum Jülich Jülich Germany

Institute of Crystallography RWTH Aachen University Aachen Germany

Institute of Physics Faculty of Science University of South Bohemia České Budějovice Czech Republic

JuStruct Jülich Center for Structural Biology Forschungszentrum Jülich Jülich Germany

M M Shemyakin and Yu A Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences Moscow Russia

N N Semenov Federal Research Center for Chemical Physics Russian Academy of Sciences Moscow Russia

Research Center for Molecular Mechanisms of Aging and Age Related Diseases Moscow Institute of Physics and Technology Dolgoprudny Russia

Technische Universität Berlin Institute of Chemistry PC14 Berlin Germany

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