Light-induced intracellular hydrogen peroxide generation through genetically encoded photosensitizer KillerRed-SOD1

Hydrogen peroxide (H₂O₂) plays an important role in various biological processes in numerous organisms. Depending on the concentration and the distribution within the cell, it can act as stressor or redox signalling molecule. To analyse the effects of H₂O₂ and its diffusion within the cell we developed the new genetically encoded photosensitizer KillerRed-SOD1 which enables a light-induced spatially and temporally controlled generation of H₂O₂ in living cells. The KillerRed-SOD1 is a fusion protein of the photosensitizer KillerRed (KR) and the cytosolic superoxide dismutase isoform 1 (SOD1) connected by a helix-forming peptide linker. Light irradiation at a wavelength of 560 nm induced superoxide radical formation at the KR domain which was transformed to H₂O₂ at the SOD1 domain. H₂O₂ was specifically detected under live cell conditions using the fluorescent sensor protein HyPer. Genetically encoded photosensitizers have the advantage that appropriate tag sequences can determine the localisation of the protein within the cell. Herein, it was exemplarily shown that the peroxisomal targeting sequence 1 directed the photosensitizer KR-SOD1 to the peroxisomes and enabled H₂O₂ formation specifically in these organelles. In summary, with the photosensitizer KR-SOD1 a new valuable tool was established which allows a controlled intracellular H₂O₂ generation for the analysis of H₂O₂ effects on a subcellular level.