Resolving sub-population dynamics in a promiscuous enzyme active site using tethered substrate analogs and 2D IR spectroscopy

Protein motion is central to enzymatic catalysis but the influence of femtosecond – picosecond timescale fluctuations on chemical reaction steps remains poorly understood. One barrier to uniting experiment and theory is difficulty in resolving the dynamics of configurational sub-populations in an ensemble. Here we use ultrafast two-dimensional infrared (2D IR) spectroscopy to examine the fluctuations about a vibrationally labeled substrate analog linked to the active site of Pyrococcus horikoshii ene-reductase (PhENR) in two orientations mimicking proposed reactive and inactive reactant states. Frequency fluctuation correlation functions (FFCFs) derived from 2D IR experiments show a near-quantitative tradeoff between fast (<1 ps) and slow (>5 ps) motions upon rotation of the analog. Increased dynamical heterogeneity and a unique ~10 cm-1 oscillation are also observed in the putative reactive configuration. These observations suggest divergent dynamics among distinct reactant state sub-populations and establish PhENR as a useful model system for continued studies.