Enzyme catalysis achieves tremendous rate accelerations. Enzyme reaction centers provide a constraint geometry that preferentially binds an activated form of the substrate and thus lowers the energy barrier. However, this transition state picture neglects the flexibility of proteins and its role in enzymatic catalysis. Especially for proton transfer reactions, it has been suggested that motions of the protein modulate the donor−acceptor distance and prepare a tunneling-ready state. We report the detection of frequency fluctuations of an azide anion (N 3 − ) bound in the active site of the protein carbonic anhydrase II, where a lowfrequency mode of the protein has been proposed to facilitate proton transfer over two water molecules during the catalyzed reaction. 2D-IR spectroscopy resolves an underdamped lowfrequency mode at about 1 THz (30 cm −1 ). We find its frequency to be viscosity-and temperature-dependent and to decrease by 6 cm −1 between 230 and 320 K, reporting the softening of the mode's potential.E nzymatic catalysis provides gains in turnover rate of up to 10 20 compared to the uncatalyzed reaction. 1 Despite the