Singlet oxygen photosensitisation by the fluorescent protein Pp2FbFP L30M, a novel derivative of Pseudomonas putida flavin-binding Pp2FbFP

Flavin-binding fluorescent proteins (FbFPs) are a class of fluorescent reporters that have been increasingly used as reporters in the study of cellular structures and dynamics. Flavin's intrinsic high singlet oxygen (¹O₂) quantum yield (Φ_Δ = 0.51) provides a basis for the development of new FbFP mutants capable of photosensitising ¹O₂ for mechanistic and therapeutic applications, as recently exemplified by the FbFP miniSOG. In the present work we report an investigation on the ¹O₂ photoproduction by Pp2FbFP L30M, a novel derivative of Pseudomonas putida Pp2FbFP. Direct detection of ¹O₂ through its phosphorescence at 1275 nm yielded the value Φ_Δ = 0.09 ± 0.01, which is the highest ¹O₂ quantum yield reported to date for any FP and is approximately 3-fold higher than the Φ_Δ for miniSOG. Unlike miniSOG, transient absorption measurements revealed the existence of two independent triplet states each with a different ability to sensitise ¹O₂.