Peroxidase activity enhancement of myoglobin by two cooperative distal histidines and a channel to the heme pocket

•The peroxidase activity of myoglobin was regulated by redesign of its heme center.•Double distal histidines in the heme pocket work cooperatively in peroxidase reactivity.•Removal of distal His64 gate forms a channel to the heme pocket facilitating substrate binding.•This study elucidates the structure and peroxidase activity relationship of heme proteins.•This study provides clues for design of heme proteins with improved catalytic performance.

To reveal the structure-function relationship of heme proteins, and to provide clues for creating artificial heme proteins with improved functions, we here use myoglobin (Mb) as a model protein, and report that its peroxidase activity can be enhanced by construction of two distal histidines and a channel to the heme pocket. It showed that in addition to a single distal histidine with a suitable distance to the heme iron (Phe43 to His43 mutation), a second distal histidine (Leu29 to His29 mutation) can work cooperatively to increase the turnover number, mimicking the role of well-known His-Arg pair in native peroxidases. Moreover, a channel created to the heme pocket by removal of the native His64 gate (His64 to Ala64 mutation) was shown to facilitate the binding of substrate, resulting in enhanced catalytic efficiency for the triple mutant L29H/F43H/H64A Mb, which is beyond the addition of both double mutants, L29H/H64A Mb and F43H/H64A Mb. These results provide valuable information for elucidating the structure-function relationship of heme proteins. In addition, this study provides clues for design of artificial heme proteins, and the strategy of creating a channel to the heme active center is expected be extended to design of other artificial enzymes with improved catalytic performance.

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