Light-induced infrared difference spectroscopy on three different forms of orange carotenoid protein: focus on carotenoid vibrations.

Orange carotenoid protein (OCP) is a photoactive carotenoprotein involved in photoprotection of cyanobacteria, which uses a keto-catorenoid as a chromophore. When it absorbs blue-green light, it converts from an inactive OCP O orange form to an activated OCP R red form, the latter being able to bind the light-harvesting complexes facilitating thermal dissipation of the excess of absorbed light energy. Several research groups have focused their attention on the photoactivation mechanism, characterized by several steps, involving both carotenoid photophysics and protein conformational changes. Among the used techniques, time-resolved IR spectroscopy have the advantage of providing simultaneously information on both the chromophore and the protein, giving thereby the possibility to explore links between carotenoid dynamics and protein dynamics, leading to a better understanding of the mechanism. However, an appropriate interpretation of data requires previous assignment of marker IR bands, for both the carotenoid and the protein. To date, some assignments have concerned specific α-helices of the OCP backbone, but no specific marker band for the carotenoid was identified on solid ground. This paper provides evidence for the assignment of putative marker bands for three carotenoids bound in three different OCPs: 3'-hydroxyechineone (3'-hECN), echinenone (ECN), canthaxanthin (CAN). Light-induced FTIR difference spectra were recorded in H 2 O and D 2 O and compared with spectra of isolated carotenoids. The use of DFT calculations allowed to propose a description for the vibrations responsible of several IR bands. Interestingly, most bands are located at the same wavenumber for the three kinds of OCPs suggesting that the conformation of the three carotenoids is the same in the red and in the orange form. These results are discussed in the framework of recent time-resolved IR studies on OCP.