Dynamical interactions between an enzyme’s substrate, cofactors, and the surrounding active site are likely to play a role in defining its catalytic activity across a broad range of timescales. In the hyperthermophilic ene-reductase from Pyrococcus horikoshii (PhENR), unsaturated substrates in close association with a flavin isoalloxazine ring system are reduced via a coupled two-proton, two-electron mechanism. To understand the role that active site dynamics play in this process, we have prepared engineered variants of PhENR containing covalently-bound substrate analogs that form substrate-like π-stacking interactions with the native flavin cofactor, observed via steady-state UV-vis spectroscopy of the flavin and x-ray crystallography. We observe enhanced inhomogeneous lineshape contributions in both the FTIR and 2D IR spectra of substrate analogs substituted with unique vibrational chromophores, including nitriles and azides, upon incorporation into the anisotropic active site environment. Modulation of the 2D IR spectral profiles of the labels incorporated into the active site and 2D IR waiting time measurements are used to characterize the contributions of label-cofactor association to catalytically-relevant dynamics on picosecond timescales.

Dynamics of a π-stacked substrate analog in a flavoenzyme active site using 2D IR spectroscopy

255th National Meeting (2018)

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