Fast Reaction of the Prussian Blue Based Nanozyme “Artificial Peroxidase” with the Substrates: Pre-Steady-State Kinetic Approach

This letter introduces the pre-steady-state kinetic approach, which is traditional for evaluation of elementary constants in molecular (enzyme) catalysis, for nanozymes. Apparently, the most active peroxidase-mimicking nanozyme based on catalytically synthesized Prussian Blue nanoparticles has been chosen. The elementary constants (<i>k</i>₁) for the nanozymes' reduction by an electron-donor substrate (being the fastest stage according to steady-state kinetic data) have been determined by means of stopped-flow spectroscopy. These constants have been found to be dependent on both the size of the nanozyme and the reducing substrate redox potential. For the smallest nanozymes (32 nm in diameter), log(<i>k</i>₁) linearly decays with an increase of the substrate redox potential (cotangent value ≈125 mV). On the contrary, for the largest nanozymes with a diameter above 150 nm, <i>k</i>₁ is almost independent of it. Moreover, for the substrate with the lowest redox potential (K₄[Fe(CN)₆]), the rate constant under discussion (<i>k</i>₁) is almost independent of the nanozymes' size. Perhaps, the rate of the intrananozyme electron transfer causing bleaching becomes comparative or even lower than that of the nanoparticle interaction with the fastest substrate. Anyway, the elementary constant of nanozyme reduction with potassium ferrocyanide (<i>k</i>₁) reaches the value of 1 × 10¹⁰ M^-1 s^-1, which is 3-4 orders of magnitude faster than for enzymes peroxidases. The obtained results obviously demonstrate that the pre-steady-state kinetic approach is able to discover novel advantages of nanozymes from both fundamental and practical points of view.