Singlet oxygen-mediated photochemical cross-linking of an engineered fluorescent flavoprotein iLOV

Genetically encoded photoactive proteins are integral tools in modern biochemical and molecular biological research. Within this tool box, truncated variants of the phototropin two light-oxygen-voltage flavoprotein have been developed to photochemically generate singlet oxygen (¹O₂) in vitro and in vivo, yet the effect of ¹O₂ on these genetically encoded photosensitizers remains underexplored. In this study, we demonstrate that the "improved" light-oxygen-voltage flavoprotein is capable of photochemical ¹O₂ generation. Once generated, ¹O₂ induces protein oligomerization via covalent cross-linking. The molecular targets of protein oligomerization by cross-linking are not endogenous tryptophans or tyrosines, but rather primarily histidines. Substitution of surface-exposed histidines for serine or glycine residues effectively eliminates protein cross-linking. When used in biochemical applications, such protein-protein cross-links may interfere with native biological responses to ¹O₂, which can be ameliorated by substitution of the surface exposed histidines of improved" light-oxygen-voltage or other ¹O₂-generating flavoproteins.