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 H 2 O 2 activation. To reveal the active centers, reaction intermediates, and dynamic structural transformations during catalysis, we investigated Fe-doped TiO 2 (Fe-TiO 2 ) nanozymes that exhibit a dual-mechanism pathway. Operando ambient-pressure electron spin resonance spectroscopy and Raman measurements revealed that H 2 O 2 molecules adsorb onto Fe-TiO 2 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 H 2 O 2, enhancing peroxidase-like activity through interactions between substrates and Ti–OOH intermediates. The inhibitory effect of l -cysteine on the activity of Fe-TiO 2 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 H 2 O 2 activation pathways mimicking natural peroxidases.