Work

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

Year: 2026

Type: preprint

Abstract: 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.

Source: bioRxiv (Cold Spring Harbor Laboratory)

DOI: 10.64898/2026.10.03.756395