What Drives the Redox Properties of Model Green Fluorescence Protein Chromophores?

T unique properties of GFP exploited in novel bioimaging techniques have revolutionized many areas in the life sciences and have motivated numerous experimental and theoretical studies. 7 To better understand the rich and complex photophysics of GFP, one can analyze its properties from bottom up, that is, from understanding the intrinsic properties of isolated FP chromophores to their behavior in the protein environment. Following this so-called reductionist approach, several studies have investigated model chromophores in the gas phase 10 and solutions. 15 Driven by mechanistic questions relevant to the GFP photocycle, these studies focused on optical properties of the chromophores, groundand excitedstate cis trans isomerization, and acidity. A recent discovery that GFP can act as a light-induced donor of electrons has brought the redox properties of FPs into the spotlight. In stark contrast with their optical properties, relatively little is known about the electron-detached (or ionized) states of FPs and, consequently, their redox properties. The gas-phase energetics of electron detachment of the anionic form of a model GFP chromophore, the deprotonated HBDI anion, has been characterized by electronic structure calculations, and, subsequently, determined experimentally. The effect ofmicrosolvation and protein environment on detachment energy (DE) has been investigated computationally. The protein matrix increases vertical DE (VDE) substantially (from 2.5 to 5.0 eV) via H-bonding and electrostatic interactions. Eox 0 of the enhanced GFP mutant (EGFP) was estimated to be 0.47 V. Experimentally, the production of solvated electrons and aromatic cation radicals has been observed for HBDI and its derivatives in solutions using femtosecond transient absorption. Interestingly, the production of solvated electrons for the meta isomer in aqueous solution was much less efficient than that for p-HBDI. It was suggested that an ionization pathway may exist in the proteinbound GFP chromophore and that the transient absorption at 1.97 to 2.48 eV observed in the pump probe experiment might be due to solvated electrons rather than excited-state absorption. Redox potentials of the so-called redox-sensitive GFPs, which change fluorescence in response to oxidation of surface cysteine groups, have been reported (thus, the measured potentials correspond to the cysteine oxidation and not the chromophore). Another study reported reduction potential of the chromophore (resulting in the nonfluorescent form) in wtGFP. Therefore, our study presents the first experimental characterization of the redox properties of model GFP chromophores in solution, which is a prerequisite for understanding the electron-donating properties of proteinbound chromophores. Knowledge of the redox potentials is important for developing generically encoded fluorescent redox probes, understanding their phototoxicity and using these chromophores in solar cell applications. In addition to