Chromophore photoreduction in red fluorescent proteins is responsible for bleaching and phototoxicity
Red fluorescent proteins (RFPs) are indispensable tools for deep tissue imaging, fluorescence resonance energy transfer (FRET), and super-resolution microscopy. Using time-resolved optical spectroscopy we investigate photoinduced dynamics of three RFPs, KillerRed, mRFP, and DsRed. In all three RFPs, we observe a new transient absorption intermediate which is formed on sub-microsecond timescale and decays on micros-ms timescale. This intermediate is characterized by red-shifted absorption at 1.68-1.72 eV (lambdamax=720-740 nm). Based on electronic structure calculations, experimental evidence, and published literature, the chemical nature of the intermediate is assigned to an unusal open-shell dianionic chromophore (dianion-radical) formed via photoreduction. Doubly charged state that is not stable for the isolated (gas-phase) chromophore is stabilized by the electrostatic field of the protein. Mechanistic implications for photobleaching, blinking, and phototoxicity are discussed.