To evaluate the peroxidase activity, we used 3,3′,5,5′-Tetramethylbenzidine (TMB) as a substrate and determined kinetic profiles using UV-vis spectroscopy. By monitoring the increase at 652 nm, we determined the rate of formation of the one-electron oxidized product (TMB + ), Figure . Furthermore, we tracked the formation of the second oxidized product (TMB 2+ ) by monitoring the increase of the 460 nm absorption band. We focused on the rate of formation of TMB + as readout of peroxidase activity. Our findings revealed that Biot2 ─ FePP effectively oxidizes TMB to TMB + , as displayed in Figure , followed by the formation of TMB 2+ in the presence of H2O2 at pH 6 in phosphate buffer. The resting ferric porphyrin reacts with H2O2 to form a high-valent intermediate, which then participates in the peroxidase-like mechanism to yield the oxidized product(s). Importantly, we excluded an alternative mechanism in which TMB is oxidized by hydroxyl radicals via Fenton chemistry due to the cofactor's reduction potential < +100 mV (vs. NHE), which maintains the Fe(III) resting state (Figure ). The UV-Vis supports the assignment of the ferric oxidation state (Figure ). Furthermore, the sequential oxidation of TMB to initially form TMB + and then TMB 2+ can be rationalized by the high concentration of the substrate (TMB), and the higher oxidation potential required to form TMB 2+ from TMB + (E TMB/TMB + 0 = +160 mV and E TMB + /TMB 2+ 0 = +300 mV vs. Ag/AgCl). However, when the reaction surface is saturated with TMB + , the high valent intermediate oxidizes it to form TMB 2+ . This again supports the involvement of a high-valent intermediate during the oxidation process, allowing it to oxidize a range of substrates with varying oxidation potentials. Additionally, the use of a pH 6 phosphate buffer in this study is noteworthy because it is a commonly used buffer for peroxidase assays and provides an optimal pH range for the enzymatic reaction, Figure .