Flavin mononucleotide (FMN) belongs to the group of very efficient endogenous photosensitizers producing singlet oxygen, O-1(2), but with limited ability to be targeted. On the other hand, in geneticallyencoded photosensitizers, which can be targeted by means of various tags, the efficiency of FMN to produce O-1(2) is significantly diminished due to its interactions with surrounding amino acid residues. Recently, an increase of O-1(2) production yield by FMN buried in a protein matrix was achieved by a decrease of quenching of the cofactor excited states by weakening of the protein-FMN interactions while still forming a complex. Here, we suggest an alternative approach which relies on the blue light irradiation-induced dissociation of FMN to solvent. This dissociation unlocks the full capacity of FMN as O-1(2) producer. Our suggestion is based on the study of an irradiation effect on two variants of the LOV2 domain from Avena sativa; wild type, AsLOV2 wt, and the variant with a replaced cysteine residue, AsLOV2 C450A. We detected irradiation-induced conformational changes as well as oxidation of several amino acids in both AsLOV2 variants. Detailed analysis of these observations indicates that irradiationinduced increase in O-1(2) production is caused by a release of FMN from the protein. Moreover, an increased FMN dissociation from AsLOV2 wt in comparison with AsLOV2 C450A points to a role of C450 oxidation in repelling the cofactor from the protein.