Ambient-Condition Visualization of Metalloenzyme Reactive Intermediates Enabled by the SHINERS Paradigm

Reactive metal–oxygen intermediates are central to metalloenzyme catalysis, yet their low populations and short lifetimes make vibrational characterization under aqueous reaction conditions challenging. Here, we establish shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) as a powerful vibrational approach for visualizing reaction-associated metal–ligand signatures of reactive metalloenzyme states. Across the heme and non-heme iron enzymes TauD, VioC, CYP119, and TqaL _Nc , isotope-sensitive features provide vibrational information on reactive coordination states. In TauD and VioC, isotope-sensitive features provide vibrational characterization of Fe(IV)=O intermediates. In CYP119, an isotope-sensitive Fe(IV)=O vibration is assigned to P450 Compound I generated by m -CPBA, including under lauric-acid turnover conditions. Most notably, in TqaL _Nc , combined ¹⁸ O- and ¹⁵ N-sensitive vibrational responses support formation of the proposed (R)HN–Fe(IV)=O coordination state and the dynamic-coordination mechanism underlying selective C–N bond formation. These results demonstrate that SHINERS can serve as a powerful technique for the in situ vibrational visualization of reactive intermediates and their coordination states in metalloenzyme catalysis.