A Genetically Encoded Two‐Dimensional Infrared Probe for Enzyme Active‐Site Dynamics.

While two‐dimensional infrared (2D‐IR) spectroscopy is uniquely suitable for monitoring femtosecond (fs) to picosecond (ps) water dynamics around static protein structures, its utility for probing enzyme active‐site dynamics is limited due to the lack of site‐specific 2D‐IR probes. We demonstrate the genetic incorporation of a novel 2D‐IR probe, m‐azido‐L‐tyrosine (N3Y) in the active‐site of DddK, an iron‐dependent enzyme that catalyzes the conversion of dimethylsulfoniopropionate to dimethylsulphide. Our results show that both the oxidation of active‐site iron to FeIII, and the addition of denaturation reagents, result in significant decrease in enzyme activity and active‐site water motion confinement. As tyrosine residues play important roles, including as general acids and bases, and electron transfer agents in many key enzymes, the genetically encoded 2D‐IR probe N3Y should be broadly applicable to investigate how the enzyme active‐site motions at the fs–ps time scale direct reaction pathways to facilitating specific chemical reactions. [ABSTRACT FROM AUTHOR]