The photoexcited triplet state of the thiouredopyrenetrisulfonate (TUPS) covalently linked to (1) alpha-acetyl lysine ((3)*TUPS-L) and (2) lysine residue 122 of azurin ((3)*TUPS-A) were studied by time-resolved EPR spectroscopy. The triplet spectra of (3)*TUPS-L and 3*TUPS-A dissolved in water-glycerol (1:I) mixture, at freezing temperatures, show different temperature dependence. While in the case of (3)*TUPS-L, no apparent dynamic effects were noticed, (3)*TUPS-A exhibits a strong effect at 230 K. The dynamic effects were treated by line shape analysis with the discrete jumps model, suggesting that the environment in which (3)*TUPS-A is embedded is not frozen, allowing for fast molecular motion with a correlation time of similar to 1.8 x 10(-10) s. These results are explained by the unique properties of the protein-bound water layers, which are kept in the liquid state even at relatively low temperatures, where the bulk water is frozen.