Fluorescence and Excited-State Conformational Dynamics of the Orange Carotenoid Protein

Abstract

The orange carotenoid protein (OCP) mediates nonphotochemical quenching (NPQ) mechanisms in cyanobacteria. A bound ketocarotenoid serves as a sensor of midvisible light intensity and as a quencher of phycocyanobilin excitons in the phycobilisome. The photochemical mechanism that triggers conversion of the protein from a resting, orange state (OCP) to an active, red state (OCP) after optical preparation of the S state of the carotenoid remains an open question. We report here that the fluorescence spectrum and quantum yield of the bound carotenoids in OCP report important details of the motions that follow optical preparation of the S state. The fluorescence spectra from OCP preparations containing 3'-hydroxyechinenone (3hECN) or canthaxanthin (CAN) are markedly mirror asymmetric with respect to the absorption line shape and more than an order of magnitude more intense than for carotenoids in solution. Further, 3hECN exhibits a narrower fluorescence line shape and a larger quantum yield than CAN because its excited-state motions are hindered by a hydrogen bonding interaction between the 3'-hydroxyl group on its β2 ring and Leu37 in the N-terminal domain. These results show that large-amplitude motions of the carotenoid's β2-cyclohexene ring and of the conjugated polyene backbone initiate photochemistry in OCP.

Excited‐State Evolution of Keto‐Carotenoids after Excess Energy Excitation in the UV Region

“…The estimated quantum yields are 3.5×10 −5 and 2×10 −5 for Ctx and Rdx, respectively. The observed fluorescence maxima as well as the Stokes’ shifts are consistent with earlier reports on fluorescence of Ctx and hydroxyechinenone [6,7] . We note, however, that the blue edge of our fluorescence spectra might be distorted due to secondary inner filter effect.…”

“…The Stokes’ shift with a considerable overlap of absorption and fluorescence bands indicates that the fluorescence occurs from the optically allowed S 2 state. The S 2 fluorescence supports the trend reported in literature that the carotenoids with longer conjugation (N eff >10) exhibit fluorescence from S 2 rather than their S 1 state [7,13] . The fluorescence maxima of Ctx (555 nm) and Rdx (585 nm) follow the red shift in their absorption peak; the energy differences between absorption and fluorescence maxima remain nearly constant (∼3100 cm −1 ) for all three carotenoids (Figure 2B, 2C).…”

“…The S 2 fluorescence supports the trend reported in literature that the carotenoids with longer conjugation (N eff > 10) exhibit fluorescence from S 2 rather than their S 1 state. [7,13] The fluorescence maxima of Ctx (555 nm) and…”

“…Ubiquitous in nature, carotenoids have a number of functions in biological systems, ranging from photoprotection and light-harvesting [2,3] to coloration [4] and structure stabilization. [5] Carotenoids exhibit extremely low fluorescence quantum yield [6,7] and their fluorescence spectra depend on the carotenoid structure. The key parameter determining the spectroscopic properties of carotenoids is the conjugation length, N, giving the number of conjugated C=C bonds in their structure.…”

Spectral Signatures of Canthaxanthin Translocation in the Orange Carotenoid Protein

“…Interestingly, the referenced studies led to the conclusion that large-amplitude motions of the β2-ring contribute to the earliest events following absorption of light that leads to photoactivation to the OCP R state. 7 Protein structural details of the photoactivation mechanism have been recently described using time-resolved electronic and vibrational spectroscopy. 8 Optical excitation of the midvisible absorption band populates the S 2 state of the carotenoid followed by ultrafast internal conversion to the dark S 1 state, which is coupled to an intramolecular charge-transfer (ICT) state.…”