Fenton-Type and Poulos–Kraut Dual Mechanisms of H₂O₂ Activation over Peroxidase-Mimicking Nanozymes Identified by Operando 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. Operando ambient-pressure electron spin resonance spectroscopy and Raman measurements revealed that H2O2 molecules adsorb onto Fe-TiO2 surfaces, occupying oxygen vacancy sites (Ti-Ov-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.