Atomically Dispersed Cu Nanozyme with Intensive Ascorbate Peroxidase Mimic Activity Capable of Alleviating ROS‐Mediated Oxidation Damage

Abstract Ascorbate peroxidase (APX) as a crucial antioxidant enzyme has drawn attentions for its utilization in preventing cells from oxidative stress responses by efficiently scavenging H2O2 in plants. For eliminating the specific inactivation of natural APXs and regulating the catalytic activity, single‐atom nanozymes are considered as promising classes of alternatives with similar active sites and maximal atomic utilization efficiency to natural APXs. Herein, graphitic carbon nitride (g‐C3N4) anchored with isolated single copper atoms (Cu SAs/CN) is designed as an efficient nanozyme with intrinsic APX mimetic behavior. The engineered Cu SAs/CN exhibits comparable specific activity and kinetics to the natural APXs. Based on the density functional theory (DFT), Cu‐N4 moieties in the active center of Cu SAs/CN are determined to exert such favorable APX catalytic performance, in which the electron transfer between Cu and coordinated N atoms facilitates the activation and cleavage of the adsorbed H2O2 molecules and results in fast kinetics. The constructed Cu SAs/CN nanozyme with superior APX‐like performance and high biocompatibility can be applied for effectively protecting the H2O2‐treated cells against oxidative injury in vitro. These findings report the single‐atom nanozymes as a successful paradigm for guiding nanozymes to implement APX mimetic performance for reactive oxygen species‐related biotherapeutic.


Ascorbate peroxidase-like activity assay
The ascorbate peroxidase (APX)-like activity of the samples was verified in a scan mode by monitoring the absorption of ascorbate with H 2 O 2 by UV-Vis spectroscopy at room temperature at 290 nm. Typically, experiments were carried out in buffer solution (pH range from 3 to 11) with a total volume of 2 mL containing Cu SAs/CN (25 μg mL -1 ) and AA (0.0625 mM). Immediately after, H 2 O 2 (5 mM) was added into the above mixture solution, the absorbance was collected over time. Meanwhile, the steady-state kinetic analysis was carried out at room temperature by varying the concentrations of AsA (0-0.125 mM) at fixed initial concentrations of Cu SAs/CN (25 μg mL -1 ) and H 2 O 2 (5 mM) in PBS solution or vice versa.
The initial reaction rate (V 0 ) against substrates was calculated based on the Michaelis-Menton function 3 : where v 0 represents the initial reaction velocity, V max refers to the maximal reaction velocity, K m and [S] are the Michaelis constant and concentration of substrate, respectively. Mean values of the initial rates of three traces were employed in the calculations. The apparent kinetic parameters (K m and V max ) were further determined from the linear double-reciprocal plots 4 : The catalytic rate constant (K cat ) of the samples were defined as: where [E] represents the molar concentration of Cu in Cu SAs/CN. Besides, the APXlike specific activity (SA) of Cu SAs/CN was calculated by:

Recycling and selective tests of Cu SAs/CN as APX mimics
To explore the robustness and stability of Cu

KSCN poisoning experiments
The thiocyanate ions (SCN -) are widely known to poison the metal-centered catalytic sites. To explore the nature of the active sites of the sing-atom catalysts, the influence of SCNis also taken into account by the previously reported works. with the above solution. The absorbance was collected in real-time mode.

Electrochemical assays
The electrochemical measurements were performed on an electrochemical The adsorption energy (E ads ) is calculated according to the formula in which E X-CN is the calculated total energy of the system with various species adsorbed either on the pristine g-C 3 N 4 or the single Cu atom doped g-C 3 N 4 ; E X represents the energy of isolated adsorbates which can be H 2 O 2 molecule, oxygen atom, OH or H 2 O molecule; and E CN is the energy of the pristine g-C 3 N 4 or the single Cu atom doped g-C 3 N 4 . A more negative E ads in equation (6) implies that the adsorption is thermodynamically more favorable. For the co-adsorption modes, the co-adsorption energy (E ads ) is calculated according to the formula

Serum stability test
For serum stability, Cu SAs/CN was incubated in DMEM cell culture medium supplemented with 10% FBS, and the hydration size of Cu SAs/CN was measured at different times, respectively.

Detection of intracellular reactive oxygen species (ROS) production
The generation of the intracellular ROS was detected by the DCFH-DA assay through a confocal laser by the microscope (FV3000, Olympus buffer, and the intracellular ROS level was detected with a confocal microscope with an excitation wavelength at 488 nm.

Statistical analysis
The experimental results for the TEM, SEM images are shown as raw data without preprocessing. The measured particle sizes were presented as their average values.
The measurement for each assay was repeated in triplicate unless otherwise noted.
Error bars in each Figure represent the standard deviation.  The obtained curves in an approximately linear manner match the ping-pong mechanism where the enzyme bind with the first substrate, releasing the product before reacting with the second one. This is in good agreement with the natural APX.