Two-Step Structural Changes in Orange Carotenoid Protein Photoactivation Revealed by Time-Resolved Fourier Transform Infrared Spectroscopy

The orange carotenoid protein (OCP), which is essential in cyanobacterial photoprotection, is the first photoactive protein containing a carotenoid as an active chromophore. Static and time-resolved Fourier transform infrared (FTIR) difference spectroscopy under continuous illumination at different temperatures was applied to investigate its photoactivation mechanism. Here, we demonstrate that in the OCP, the photo-induced conformational change involves at least two different steps, both in the second timescale at 277 K. Each step involves partial reorganization of alpha-helix domains. At early illumination times, the disappearance of a nonsolvent-exposed alpha-helix (negative 1651 cm(-1) band) is observed. At longer times, a 1644 cm(-1) negative band starts to bleach, showing the disappearance of a solvent-exposed a-helix, either the N-terminal extension and/or the C-terminal tail. A kinetic analysis clearly shows that these two events are asynchronous. Minor modifications in the overall FTIR difference spectra confirm that the global protein conformational change consists of-at least-two asynchronous contributions. Comparison of spectra recorded in H2O and D2O suggests that internal water molecules may contribute to the photoactivation mechanism.