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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]

• The excited state dynamics processes for TTB and DSB. • Conversion of excited states from LE to CT with increasing solvent polarity. • Solvent polarity facilitates the hRISC process and leads to higher fluorescence quantum yield. The relationship between the photophysical properties of N,N,N',N' -tetra-p-tolyl-benzidine (TTB) and [1,4-bis[2-[4-[ N,N -di(p-tolyl)amino]phenyl]vinyl]benzene (DSB) molecules and solvent polarity was explored through steady-state spectroscopy, femtosecond transient absorption spectroscopy, and quantum chemical calculations. Computational results and steady-state spectra reveal that the lowest singlet excited states of TTB and DSB are primarily characterized by localized excited (LE) states in nonpolar cyclohexane (CHX), whereas intramolecular charge transfer (ICT) components predominate in highly polar acetonitrile (ACN). Compared to TTB, the excited-state potential of DSB demonstrates a heightened sensitivity to solvent polarity, exhibiting larger dipole moments due to enhanced electron transfer enabled by the incorporation of styrene groups. Ultrafast femtosecond transient absorption spectroscopy distinctly shows the transition from LE states in nonpolar CHX to ICT states in highly polar ACN. The deactivation mechanisms were further elucidated to understand how solvent polarity modulates excited states and high quantum yields, providing valuable insights for the design and application of photophysical materials. [ABSTRACT FROM AUTHOR]