Chromophore photoreduction in red fluorescent proteins is responsible for bleaching and phototoxicity

R.B. Vegh, K.B. Bravaya, D.A. Bloch, A.S. Bommarius, L.M. Tolbert, M. Verkhovsky, A.I. Krylov, and K.M. Solntsev
J. Phys. Chem. B 118, 4527 – 4534 (2014)

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 (lambda_max=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.

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