Chromophore Photoreduction in Red Fluorescent Proteins Is Responsible for Bleaching and Phototoxicity

[Image: see text] Red fluorescent proteins (RFPs) are indispensable tools for deep-tissue imaging, fluorescence resonance energy transfer applications, and super-resolution microscopy. Using time-resolved optical spectroscopy this study investigated photoinduced dynamics of three RFPs, KillerRed, mRFP, and DsRed. In all three RFPs, a new transient absorption intermediate was observed, which decays on a microsecond–millisecond time scale. This intermediate is characterized by red-shifted absorption at 1.68–1.72 eV (λ(max) = 720–740 nm). On the basis of electronic structure calculations, experimental evidence, and published literature, the chemical nature of the intermediate is assigned to an unusual open-shell dianionic chromophore (dianion-radical) formed via photoreduction. A doubly charged state that is not stable in the isolated (gas phase) chromophore is stabilized by the electrostatic field of the protein. Mechanistic implications for photobleaching, blinking, and phototoxicity are discussed.