Temperature Sensitive Singlet Oxygen Photosensitization by LOV-Derived Fluorescent Flavoproteins.

Optogenetic sensitizers that selectively produce a given reactive oxygen species (ROS) constitute a promising tool for studying cell signaling processes with high levels of spatiotemporal control. However, to harness the full potential of this tool for live cell studies, the photophysics of currently available systems need to be explored further and optimized. Of particular interest in this regard, are the flavoproteins miniSOG and SOPP, both of which (1) contain the chromophore flavin mononucleotide, FMN, in a LOV-derived protein enclosure, and (2) photosensitize the production of singlet oxygen, O(aΔ). Here we present an extensive experimental study of the singlet and triplet state photophysics of FMN in SOPP and miniSOG over a physiologically relevant temperature range. Although changes in temperature only affect the singlet excited state photophysics slightly, the processes that influence the deactivation of the triplet excited state are more sensitive to temperature. Most notably, for both proteins, the rate constant for quenching of FMN by ground state oxygen, O(XΣ), increases ∼10-fold upon increasing the temperature from 10 to 43 °C, while the oxygen-independent channels of triplet state deactivation are less affected. As a consequence, this increase in temperature results in higher yields of O(aΔ) formation for both SOPP and miniSOG. We also show that the quantum yields of O(aΔ) production by both miniSOG and SOPP are mainly limited by the fraction of FMN triplet states quenched by O(XΣ). The results presented herein provide a much-needed quantitative framework that will facilitate the future development of optogenetic ROS sensitizers.