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Protein design has received much attention in the last decades. With an additional disulfide bond to enhance the protein stability, human A15C neuroglobin (Ngb) is an ideal protein scaffold for heme enzyme design. In this study, we rationally converted A15C Ngb into a multifunctional peroxidase by replacing the heme axial His64 with an Asp residue, where Asp64 and the native Lys67 at the heme distal site were proposed to act as an acid-base catalytic couple for H2O2 activation. Kinetic studies showed that the catalytic efficiency of A15C/H64D Ngb was much higher (∼50-80-fold) than that of native dehaloperoxidase, which even exceeds (∼3-fold) that of the most efficient native horseradish peroxidase. Moreover, the dye-decolorizing peroxidase activity was also comparable to that of some native enzymes. Electron paramagnetic resonance, molecular docking, and isothermal titration calorimetry studies provided valuable information for the substrate-protein interactions. Therefore, this study presents the rational design of an efficient multifunctional peroxidase based on Ngb with potential applications such as in bioremediation for environmental sustainability.

The function of the highly conserved residue Asn52 in human cytochrome c (H-Cyt c) is not fully understood. Herein, we show that the naturally occurring variant N52S H-Cyt c has a perturbed secondary structure, with a small fraction of high-spin species. Remarkably, it exhibits an enhanced peroxidase activity by 3–8-fold at neutral pH, as well as self-oxidation in reaction with H2O2. This study suggests that the H-bond network mediated by Asn52 is essential to suppress the apoptotic activity of H-Cyt c under physiological conditions.