Abstract
The 35-kDa Orange Carotenoid Protein (OCP) is responsible for photoprotection in cyanobacteria. It acts as a light intensity sensor and efficient quencher of phycobilisome excitation. Photoactivation triggers large-scale conformational rearrangements to convert OCP from the orange OCPO state to the red active signaling state, OCPR, as demonstrated by various structural methods. Such rearrangements imply a complete, yet reversible separation of structural domains and translocation of the carotenoid. Recently, dynamic crystallography of OCPO suggested the existence of photocycle intermediates with small-scale rearrangements that may trigger further transitions. In this study, we took advantage of single 7 ns laser pulses to study carotenoid absorption transients in OCP on the time-scale from 100 ns to 10 s, which allowed us to detect a red intermediate state preceding the red signaling state, OCPR. In addition, time-resolved fluorescence spectroscopy and the assignment of carotenoid-induced quenching of different tryptophan residues derived thereof revealed a novel orange intermediate state, which appears during the relaxation of photoactivated OCPR to OCPO. Our results show asynchronous changes between the carotenoid- and protein-associated kinetic components in a refined mechanistic model of the OCP photocycle, but also introduce new kinetic signatures for future studies of OCP photoactivity and photoprotection.
Highlights
The 35-kDa Orange Carotenoid Protein (OCP) utilizes the extraordinary capability of its carotenoid cofactor to convert electronic excitation energy by internal conversion within a few picoseconds into harmless heat in order to shield the sensitive photosystems from excessive excitation energy arriving from the phycobilisome (PBs) antennae complexes at high light intensities[1,2,3,4,5,6,7,8,9]
Binding of Fluorescence Recovery Protein (FRP) occurs to the C-terminal domain (CTD), most likely at a site covered in OCPO by the αA-helix, some secondary, as yet unidentified, site might exist on the N-terminal domain (NTD) or the interdomain linker too, in order to enable the reversion of domain separation and back-sliding of the carotenoid cofactor into its initial position[22,32]
Our transient absorption experiments (Fig. 1) show that after irradiation of the sample with a 7-ns laser flash, the increase of OCP absorption at 550 nm occurs faster than 200 ns, which is the time resolution of our instrument. This remarkable feature indicates that only small changes of chromophore–protein interactions are necessary for the spectral conversion of OCPO to a state with red-shifted chromophore absorption
Summary
The 35-kDa Orange Carotenoid Protein (OCP) utilizes the extraordinary capability of its carotenoid cofactor to convert electronic excitation energy by internal conversion within a few picoseconds into harmless heat in order to shield the sensitive photosystems from excessive excitation energy arriving from the phycobilisome (PBs) antennae complexes at high light intensities[1,2,3,4,5,6,7,8,9]. According to biochemical (size-exclusion chromatography, SEC) and structural (small-angle X-ray scattering, SAXS, or static and dynamic X-ray crystallography) studies, the active, quenching OCPR state is characterized by increased disorder in terms of a molten globule, detachment and unfolding of the αA-helix from the CTD, domain separation, and, eventually, a carotenoid translocation by 12 Å into the NTD, thereby forming a spectral state indistinguishable from the one of the Red Carotenoid Protein (RCP) The latter is a hydrolysis product of OCP comprising only the carotenoid-bound N-terminal domain, which constitutively quenches PBs fluorescence[25,26,27,28]. This range of timescales (10−7–10−3 s) has been demonstrated to be crucial for the photocycle of other photoactive proteins[39,40,41], and frequently comprises transitions between several intermediate states
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