Intramolecular Fluorescence Quenching of Tyrosine by the Peptide α-Carbonyl Group Revisited

The environment-dependent multiexponential behavior of N-acetyltyrosinamide (NAYA) and other tyrosine derivatives is revisited, aiming for a better understanding of tyrosine as an intrinsic fluorescent probe for protein microenvironment changes during conformational changes. The effects of solvent polarity, viscosity, and temperature on the fluorescence decay of NAYA were evaluated using dioxane - Water mixtures and pure solvents. Double-exponential decays were observed in dioxane - Water mixtures above 70% (v/v) water concentration including pure water, for temperatures below 50 °C. However, at higher temperatures, or in dioxane - Water mixtures with lower water concentrations, NAYA shows single-exponential decays. Single-exponential decays were also generally observed in pure solvents (dioxane, acetonitrile, methanol, ethanol, DMSO). The exception was the strong hydrogen-bond donor trifluorethanol, in which NAYA decays as a double exponential. The results are consistent with a solvent-modulated excited-state intramolecular electron transfer from the phenol to the amide moiety occurring in one of the three rotamers of NAYA. On the basis of a full kinetic analysis of the data, it is shown that experimental observation of double-exponential decays depends on three factors: the ground-state population of rotamers, their rates of interconversion (k _r = 4.4 × 10 ⁸ s ^-1 and k _r′ = 5.2 × 10 ⁸ s ^-1, in water at 23°C, E _r = 4.9±0.1 kcal mol ^-1 and E _r′= 5.2±0.3 kcal mol ^-1), and the electron-transfer rate constant (k _ET = 2.0 × 10 ⁹ s ^-1, in water at 23°C, E _ET = 1.8±0.2 kcal mol ^-1). Solvent viscosity controls the interconversion rate constants while solvent polarity and hydrogen-bonding ability determines the Gibbs energy of electron transfer and the magnitude of its rate constant. Consequently, the nature of tyrosine decays in proteins is determined from a delicate balance between the interconversion and electron-transfer rate constants.