A Single Molecule Investigation of Pycobilisome Energy Transfer and Excess Light Protection Mechanisms

Abstract

Phycobilisomes (PBS) are light harvesting antenna complexes in cyanobacteria and red algae. A PBS harvests light energy by using hundreds of phycobilin fluorophores that are evenly distributed in PBS disk components, and the captured energy is believed to transfer to reaction centers in the thylakoid membrane via a set of terminal emitter fluorophores. Although a pair of phycobilins located within the two Lcm linker polypeptides and another pair in the core cylinders have been proposed to be the terminal emitters, their locations in PBS have not been measured. Utilizing single molecule fluorescence imaging and localization measurements, we observed two fluorescence quantum bleaching events in isolated PBS molecules attached to fused silica substrate. We identify these events as the two “terminal located within the PBS core. By measuring the separation of the two bleaching events, we determine the distance between the two terminal emitters to be 7 nm. Surprisingly, the separation of the two terminal emitters increases with the excitation light intensity from 7 nm to 20 nm, indicating that (i) the locations of terminal emitters are dynamic, and (ii) which may be a consequence of a new excess exposure protection mechanism of PBS by sterically mismatching the terminal emitters and reaction centers' energy transfer sites. Furthermore, we have observed a decrease in PBS fluorescence emission lifetime photon with increased exposure, suggesting the existence of Non-Photochemical Quenching in PBS alone without the involvement of Orange Carotenoid Proteins that is believed to be necessary for PBS' thermal dissipation of excess absorbed energy.