In cyanobacteria, the orange carotenoid protein (OCP) mediates nonphotochemical quenching of bilin excited states in the phycobilisome. An intrinsic ketocarotenoid serves in OCP as an ambient light-intensity sensor by triggering the conversion of the resting, orange form (OCPO) to the active, red form (OCPR), which can then bind to the phycobilisome's core.We have performed absorbance and fluorescence measurements to determine the process by which the carotenoid mediates photoactivation. This dissertation describes an intermediate protein species between the resting and active states, which is formed by the separation of a protein dimer unit into two separate monomers. This intermediate, herein named OCPI, converts further to form the active state, OCPR. A wavelength-dependent study of the photoactivation process reveals increased rates of photoproduct formation when exposed to higher energy actinic light. Contrary to previous literature asserting that the photoactivation process occurs due to bond length alternation, these results suggest that the carotenoid distorts through nonplanar coordinates to initiate the photoactivation process.We subsequently measured the distortion of the ketocarotenoid canthaxanthin (CAN) through fluorescence anisotropy. These experiments reveal CAN undergoes twisting and bending displacements along the isoprenoid backbone during the decay pathway from the S2 state via the SX intermediate to the dark S1 state, even when sterically constrained. In solution, the carotenoid forms a pyramidal structure with significant intramolecular charge-transfer character. However, in the protein cavity, the carotenoid distorts primarily through concerted volume-conserving motions of more than one carbon bond, such as those of the “bicycle-pedal” mechanism.These findings indicate definitively that the photoactivation process is initiated by torsional motions of the ketocarotenoid inside the OCP structure, and that these motions are not hindered by the protein’s residues. We propose that the ketocarotenoid triggers the photoactivation reaction by shortening its effective length through multiple out-of-plane distortions, thereby breaking hydrogen bonds with the protein and allowing displacement of the C-terminal domain.
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