LAUSR

Abstract The design of artificial metalloenzymes has achieved tremendous progress, although few designs can achieve catalytic performances comparable to that of native enzymes. Moreover, the structure and function of artificial metalloenzymes in non‐native states has rarely been explored. Herein, we found that a c‐type cytochrome b 5 (Cyt b 5), N57C/S71C Cyt b 5, with heme covalently attached to the protein matrix through two Cys–heme linkages, adopts a non‐native state with an open heme site after guanidine hydrochloride (Gdn⋅HCl)‐induced unfolding, which facilitates H2O2 activation and substrate binding. Stopped‐flow kinetic studies further revealed that c‐type Cyt b 5 in the non‐native state exhibited impressive peroxidase activity comparable to that of native peroxidases, such as the most efficient horseradish peroxidase. This study presents an alternative approach to the design of functional artificial metalloenzymes by exploring enzymatic functions in non‐native states.