Fenton-Type and Poulos–Kraut Dual Mechanisms of H₂O₂ Activation over Peroxidase-Mimicking Nanozymes Identified by <i>Operando</i> Measurements

Current strategies for developing peroxidase-mimicking nanozymes seldom address the interplay between Fenton-type hemolytic and Poulos-Kraut heterolytic mechanisms in H₂O₂ activation. To reveal the active centers, reaction intermediates, and dynamic structural transformations during catalysis, we investigated Fe-doped TiO₂ (Fe-TiO₂) nanozymes that exhibit a dual-mechanism pathway. <i>Operando</i> ambient-pressure electron spin resonance spectroscopy and Raman measurements revealed that H₂O₂ molecules adsorb onto Fe-TiO₂ surfaces, occupying oxygen vacancy sites (Ti-O_v-Ti) and forming peroxy bonds with Ti atoms (Ti-OOH). The incorporation of Fe facilitates both Fenton-type homolytic cleavage and Poulos-Kraut heterolytic cleavage of H₂O₂, enhancing peroxidase-like activity through interactions between substrates and Ti-OOH intermediates. The inhibitory effect of l-cysteine on the activity of Fe-TiO₂ nanozymes inspired a rapid and selective l-cysteine biosensor. This study reveals that defect engineering introduces the Poulos-Kraut mechanism into peroxidase-mimicking nanozymes as an innovative alternative to the Fenton-type mechanism, offering a promising approach for exploring dual H₂O₂ activation pathways mimicking natural peroxidases.