Anisotropic Dynamics of an Interfacial Enzyme Active Site Observed Using Tethered Substrate Analogs and Ultrafast 2D IR Spectroscopy

A number of recent reports have implicated ultrafast (femtosecond-picosecond) timescale motions in enzymatic activity, but relatively few experimental studies have addressed complications arising from spatially-distributed disorder, multiple substrate binding modes, or the influence of hydration dynamics on solvent-exposed active sites. Here we use ultrafast two-dimensional infrared (2D IR) spectroscopy and covalently-tethered substrate analogs to examine dynamical properties of the Pyrococcus horikoshii ene-reductase (PhENR) active site in two defined binding configurations. Frequency-fluctuation correlation functions of aryl-nitrile analogs reveal an end-to-end tradeoff between fast (sub-ps) and slow (>5 ps) motions. Lineshape and quantum beat analyses of Fermi resonant aryl-azide analogs demonstrate that this is an intrinsic property of the water-exposed active site. This study indicates that elements of polar pre-organization are maintained at the interface and suggests several plausible factors leading to state-selective rate enhancement and promiscuity in PhENR.