Photoinduced bleaching in an efficient singlet oxygen photosensitizing protein: Identifying a culprit in the flavin-binding LOV-based protein SOPP3

[Display omitted] •Bleaching of FMN in SOPP3 depends on electron transfer with surrounding molecules.•Amino acids in SOPP3 mediate the bleaching reaction of bound FMN.•Despite FMN bleaching, SOPP3 is still be a viable intracellular optogenetic actuator. Light-oxygen-voltage (LOV) proteins that bind flavin mononucleotide (FMN) have received attention as optogenetic systems that can photosensitize the production of singlet molecular oxygen, O2(a1Δg). Although the seminal compound in this family, mini singlet oxygen generator (miniSOG), does not make O2(a1Δg) in appreciable yield, it is a convenient standard against which other singlet oxygen photosensitizing proteins (SOPPs) can be judged. SOPP3 is a re-engineered version of miniSOG that selectively produces O2(a1Δg) in high yield but, when localized in a cell, the protein-encased FMN readily bleaches upon irradiation. In contrast, the bleaching of FMN in miniSOG is slow under the same conditions. The available evidence indicates that O2(a1Δg) is not directly involved in the bleaching reaction. Rather, electron-transfer reactions between FMN and molecules in the surrounding medium, likely mediated by residues in the LOV protein, accelerate FMN bleaching. In this regard, and identifying a culprit, replacing a glutamine that H-bonds to FMN in miniSOG with either leucine (Q103L) or valine (Q103V) increases the 3FMN lifetime and, thereby, increases both the O2(a1Δg) yield and the bleaching rate. Although the same electron transfer reactions likely influence these respective observations, the O2(a1Δg) yield principally reflects the kinetic competition between two processes that remove/deactivate 3FMN: (1) quenching by ground state oxygen, O2(X3Σg-), to produce O2(a1Δg), and (2) electron transfer to produce the FMN radical anion. Photobleaching principally reflects the accumulated electron-transfer-mediated depletion of ground state FMN, which, over time in the dark, is partly regenerated by compensating redox reactions. Despite the bleaching, irradiation of SOPP3 induces morphological change in a cell. This result, which contradicts conclusions from previous experiments with a lower light dose, indicates that SOPP3 may indeed be a viable optogenetic actuator.