Flavin mononucleotide (FMN) belongs to the group of very efficient endogenous photosensitizers producing singlet oxygen, 1 o 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 1 o 2 is significantly diminished due to its interactions with surrounding amino acid residues. Recently, an increase of 1 o 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 1 o 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 1 o 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. Flavin mononucleotide (FMN) belongs to a group of efficient endogenous photosensitizers in cells with rather high singlet oxygen, 1 O 2 , quantum yield (Φ Δ) within the range 0.51-0.65 1,2. Depending on FMN concentrations and concentrations of available oxygen, the flavin(s) can be even more effective 1 O 2 generators than exogenous porphyrins used for cell killing in photodynamic therapy (PDT). To minimize the potential deleterious effect of flavins to cells, the isoalloxazine moiety of flavin cofactors is typically deeply buried in the protein core of flavoenzymes 3 or storage proteins 4. Singlet oxygen, the lowest energy excited electronic state of molecular oxygen, belongs to the group of reactive oxygen species (ROS), which includes superoxide anion (O 2 •−), hydrogen peroxide (H 2 O 2), and hydroxyl radical (HO •), enabling to oxidize and/or oxygenate many biologically relevant molecules 5,6. Singlet oxygen can be produced in a variety of ways by physical mechanisms, including energy transfer from the excited triplet states of particular chromophores to molecular oxygen 7 , or by chemical mechanisms as one of the products of peroxidase enzymes 8. In biological systems, 1 O 2 is usually generated by electronic energy transfer from an excited state of a photosensitive molecule, so-called photosensitizer (PS), to ground state O 2 6. The high reactivity of singlet oxygen towards biological molecules is relevant in the context of PDT 9 and chromophore-assisted laser inactivation (CALI) of proteins and cells 10,11 .