miniSOG, developed as the first fully genetically encoded
singlet oxygen photosensitiser, has found various applications in cell imaging and functional studies. Yet, miniSOG has suboptimal properties, including a low yield of
singlet oxygen generation, which can nevertheless be improved tenfold upon blue light irradiation. In a previous study, we showed that this improvement was due to the photolysis of the miniSOG chromophore,
flavin mononucleotide (
FMN), into
lumichrome, with concomitant removal of the phosphoribityl tail, thereby improving
oxygen access to the
alloxazine ring. We thus reasoned that a chromophore with a shorter tail would readily improve the photosensitizing properties of miniSOG. In this work, we show that the replacement of
FMN by
riboflavin (RF), which lacks the bulky
phosphate group, significantly improves the
singlet oxygen quantum yield (ΦΔ). We then proceeded to mutagenize the residues stabilizing the
phosphate group of
FMN to alter the chromophore specificity. We identified miniSOG-R57Q as a
flavoprotein that selectively binds RF in cellulo, with a modestly improved ΦΔ. Our results show that it is possible to modify the
flavin specificity of a given
flavoprotein, thus providing a new option to tune its photophysical properties, including those leading to
photosensitization. We also determined the structure of miniSOG-Q103L, a mutant with a much increased ΦΔ, which allowed us to postulate the existence of another access channel to
FMN for molecular
oxygen.