UC Santa Barbara

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.