experimental details , xray crystal structure of culoaded f33y cubmb ,   malditof mass spectra , epr simulation , and extra uv / vis , epr spectra are provided in the supporting information .
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 abstractrational protein design has been proven to be a powerful tool for creating functional artificial proteins . 
 although many artificial metalloproteins with a single active site have been successfully created , those with dual active sites in a single protein scaffold are still relatively rare . in this study , we rationally designed dual active sites in a single heme protein scaffold , myoglobin ( mb ) , by retaining the native heme site and creating a copperbinding site remotely through a single mutation of arg118 to his or met . 
 isothermal titration calorimetry ( itc ) and electron paramagnetic resonance ( epr ) studies confirmed that a copperbinding site of [ 3his ] or [ 2his1met ] motif was successfully created in the single mutant of r118h mb and r118 m mb , respectively . 
 uv / vis kinetic spectroscopy and epr studies further revealed that both the heme site and the designed copper site exhibited nitrite reductase activity . 
 this study presents a new example for rational protein design with multiple active sites in a single protein scaffold , which also lays the groundwork for further investigation of the structure and function relationship of heme / nonheme proteins . 
Experimental Section
Supporting information