Electric Field Effects in the Active Site of a Thermophilic Enzyme as Observed by FTIR and 2D IR Spectroscopy

Our research aims to understand how changes in ultrafast dynamics compare and correlate to thermophilic enzyme activity. We observe fluctuating electric field effects in a promiscuous, hyperthermophilic ene-reductase from Pyrococcus horikoshii (PhENR) to address this. This enzyme catalyzes the reduction of activated alkenes/alkynes to their respective alkanes/alkenes via proton and hydride transfers from a flavin cofactor in the active site. We exploit the promiscuity of PhENR in order to incorporate a variety of substrates and substrate analogs into the active site for these studies. We have synthesized a set of covalently-attached substituted N-phenylmaleimide infrared labels, which mimic the structures of the enzyme's substrates, and contain unique vibrational chromophores to probe the enzyme's active site dynamics. Current studies focus on the vibrational frequencies and lineshapes of nitrile labels such as those of 4-cyano-N-phenylmaleimide, which sits proximal to the catalytic flavin and can be attached in multiple orientations within the active site. When compared to the label in solution, the covalently attached label undergoes significant inhomogeneous broadening in its FTIR spectrum reflecting the distribution of active site microenvironments. Additionally, protein-based non-natural amino acid labels such as methionine to azidohomoalanine substitutions are also being incorporated into the distal side of the flavin cofactor for similar studies in different location within the enzyme's active site. Using 2D IR spectroscopy, we are examining the contributions of femtosecond to picosecond active site dynamics to the lineshapes of both the covalently attached probes as well as the incorporated non-natural amino acid labels. Future research aims to break the thermophilicity of the enzyme via specific mutations in order to compare the active site dynamics to a corresponding mesophilic version of the protein.