The orange carotenoid protein (OCP) is a structurally and functionally modular photoactive protein involved in cyanobacterial photoprotection. Recently, based on bioinformatic analysis and phylogenetic relationships, new families of OCP have been described, OCP2 and OCPx. The first characterization of the OCP2 showed both faster photoconversion and back-conversion, and lower fluorescence quenching of phycobilisomes relative to the well-characterized OCP1. Moreover, OCP2 is not regulated by the fluorescence recovery protein (FRP). In this work, we present a comprehensive study combining ultrafast spectroscopy and structural analysis to compare the photoactivation mechanisms of OCP1 and OCP2 from Tolypothrix PCC 7601. We show that despite significant differences in their functional characteristics, the spectroscopic properties of OCP1 and OCP2 are comparable. This indicates that the OCP functionality is not directly related to the spectroscopic properties of the bound carotenoid. In addition, the structural analysis by X-ray footprinting reveals that, overall, OCP1 and OCP2 have grossly the same photoactivation mechanism. However, the OCP2 is less reactive to radiolytic labeling, suggesting that the protein is less flexible than OCP1. This observation could explain fast photoconversion of OCP2.

ELI Beamlines Institute of Physics Czech Academy of Sciences Za Radnicí 835 252 41 Dolní Břežany Czech Republic

Environmental Genomics and Systems Biology Division Lawrence Berkeley National Laboratory Berkeley CA 94720 USA

Institute of Physics Faculty of Science University of South Bohemia Branišovská 1760 370 05 České Budějovice Czech Republic

Molecular Biophysics and Integrated Bioimaging Division Lawrence Berkeley National Laboratory Berkeley CA 94720 USA

MSU DOE Plant Research Laboratory Michigan State University East Lansing MI 48824 USA

MSU DOE Plant Research Laboratory Michigan State University East Lansing MI 48824 USA; Molecular Biophysics and Integrated Bioimaging Division Lawrence Berkeley National Laboratory Berkeley CA 94720 USA; Environmental Genomics and Systems Biology Division Lawrence Berkeley National Laboratory Berkeley CA 94720 USA

MSU DOE Plant Research Laboratory Michigan State University East Lansing MI 48824 USA; Molecular Biophysics and Integrated Bioimaging Division Lawrence Berkeley National Laboratory Berkeley CA 94720 USA; Environmental Genomics and Systems Biology Division Lawrence Berkeley National Laboratory Berkeley CA 94720 USA; Department of Biochemistry and Molecular Biology Michigan State University East Lansing MI 48824 USA

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Structures of a phycobilisome in light-harvesting and photoprotected states

Role of hydrogen bond alternation and charge transfer states in photoactivation of the Orange Carotenoid Protein

Structural analysis of a new carotenoid-binding protein: the C-terminal domain homolog of the OCP