Westberg M.,Center for Oxygen Microscopy and Imaging |
Holmegaard L.,Center for Oxygen Microscopy and Imaging |
Pimenta F.M.,Center for Oxygen Microscopy and Imaging |
Etzerodt M.,University of Aarhus |
Ogilby P.R.,Center for Oxygen Microscopy and Imaging
Journal of the American Chemical Society | Year: 2015
Singlet oxygen, O2(a1Δg), 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 O2(a1Δg) production in or on a cell. As such, there is great need for a system that (a) selectively produces O2(a1Δg) 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 O2(a1Δg) production. As a consequence, our protein-encased FMN system produces O2(a1Δg) 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 O2(a1Δg) formation kinetics and yields. As such, our results facilitate future systematic developments in this field. © 2015 American Chemical Society.