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.

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Enzymes accelerate the rates of biomolecular reactions by many orders of magnitude compared to bulk solution, and it is widely understood that this catalytic effect arises from a combination of polar pre-organization and electrostatic transition state stabilization. A number of recent reports have also implicated ultrafast (femtosecond-picosecond) timescale motions in enzymatic activity. However, complications arising from spatially-distributed disorder, the occurrence of multiple substrate binding modes, and the influence of hydration dynamics on solvent-exposed active sites still confound many experimental studies. Here we use ultrafast two-dimensional infrared (2D IR) spectroscopy and covalently-tethered substrate analogs to examine dynamical properties of the promiscuous Pyrococcus horikoshii ene-reductase (PhENR) active site in two binding configurations mimicking proposed ‘inactive’ and ‘reactive’ Michaelis complexes. Spectral diffusion measurements of aryl-nitrile substrate analogs reveal an end-to-end tradeoff between fast (sub-ps) and slow (>5 ps) motions. Fermi resonant aryl-azide analogs that sense interactions of coupled oscillators are described. Lineshape and quantum beat analyses of these probes reveal characteristics that correlate with aryl-nitrile FFCF parameters, demonstrating that dynamical anisotropy is an intrinsic property of the water-exposed active site, where countervailing gradients of fast dynamics and disorder in the reactant ground state are maintained near the hydration interface. Our results suggest several plausible factors leading to state-selective rate enhancement and promiscuity in PhENR. This study also highlights a strategy to detect perturbations to vibrational modes outside the transparent window of the mid-IR spectrum, which may be extended to other macromolecular systems.

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.