MechanisticInsights into HRP-Catalyzed Oxidationof the Near-Infrared Fluorescent Dye Sulfo-cyanine 7

Horseradish peroxidase (HRP) is a widely used enzyme that oxidizes a range of substrates in the presence of its co-substrate hydrogen peroxide (H₂O₂). In the past, we identified sulfo-cyanine 7 (S7) as a novel near-infrared substrate of HRP, which loses its fluorescence upon enzymatic oxidation in a hydrogen peroxide-dependent manner. However, the catalytic mechanism and the chemical structure of the oxidation products were not well understood. In this study, we investigate the catalytic mechanism of S7 oxidation by HRP and identify two radical products formed during the reaction. Our results point to the formation of non-near-infrared fluorescent species, and the regeneration of S7. These results strongly point to a disproportionation reaction that was described for other HRP substrates, namely the structurally related ABTS (2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)).