Rational design of an efficient, genetically encodable, protein-encased singlet oxygen photosensitizer.

Singlet oxygen, O-2(a(1)Delta(g)), plays a key role in many processes of cell signaling. Limitations in mechanistic studies of such processes are generally associated with the difficulty of controlling the amount and location of O-2(a(1)Delta(g)) production in or on a cell. As such, there is great need for a system that (a) selectively produces O-2(a(1)Delta(g)) in appreciable and accurately quantifiable yields and (b) can be localized in a specific place at the suborganelle level. A genetically encodable, protein-encased photosensitizer is one way to achieve this goal. Through a systematic and rational approach involving mutations to a LOV2 protein that binds the chromophore flavin mononucleotide (FMN), we have developed a promising photosensitizer that overcomes many of the problems that affect related systems currently in use. Specifically, by decreasing the extent of hydrogen bonding between FMN and a specific amino acid residue in the local protein environment, we decrease the susceptibility of FMN to undesired photoinitiated electron-transfer reactions that kinetically compete with O-2(a(1)Delta(g)) production. As a consequence, our protein-encased FMN system produces O-2(a(1)Delta(g)) with the uniquely large quantum efficiency of 0.25 +/- 0.03. We have also quantified other key photophysical parameters that characterize this sensitizer system, including unprecedented H2O/D2O solvent isotope effects on the O-2(a(1)Delta(g)) formation kinetics and yields. As such, our results facilitate future systematic developments in this field.