Multiple catalytic functions of an engineered double mutant of myoglobin with a potential metal-binding site

Abstract

Rational protein engineering has emerged as a powerful tool for creating functional enzymes, and the design of metalloenzymes with dual active sites is particularly attractive. In this study, we performed a double mutation of F46H/L49D in the helices C and D region in myoglobin (Mb). As demonstrated by X-ray crystallography, the double mutations preserved the overall Mb fold and formed a potential metal-binding site, located ∼15 Å from the heme iron, which enable the protein to bind various metal ions such as Cu²⁺, Mg²⁺ and others. Moreover, the binding of Cu²⁺/Mg²⁺ conferred multiple enzymatic activities to F46H/L49D Mb. The Cu²⁺-F46H/L49D Mb complex exhibited significant nitrite reductase and superoxide dismutase activities. Notably, the protein exhibited DNA cleavage activity in the presence of Mg²⁺, achieving nearly 100 % cleavage efficiency within 30 min. By demonstrating the versatility of the engineered metal-binding site in Mb, this study suggests that rational design can expand the functional repertoire of the protein. The F46H/L49D Mb mutant serves as a versatile platform for studying metal-dependent catalysis of artificial metalloenzymes with non-heme/heme dual active sites, offering potential applications in biocatalysis, medicine, and industrial catalysis.