Abstact

Type 3 (T3) Cu proteins play essential roles in binding and activating molecular oxygen (O(2)) and are prevalent across all domains of life. Despite sharing the same coordination motif, T3 Cu proteins display divergent functions: hemocyanin transports O(2), while tyrosinase catalyzes the hydroxylation of monophenols and the subsequent oxidation of diphenols and catechol oxidase oxidizes only diphenols. Here, we report the design and characterization of a di-Cu protein (Cu-HC4) inspired by the active sites of natural T3 Cu proteins to investigate the structural features that facilitate catalytic oxidase activity. Cu-HC4 is roughly 1/5th the size of the commercially available mushroom tyrosinase and shares only around 20% sequence identity with the T3 Cu protein templates. Notably, Cu-HC4 displays high thermostability and exhibits diphenol oxidation activity at ambient and elevated temperatures (>/=60 degrees C). Cu-HC4 also initiates the formation of melanin polymers, mimicking melanin biosynthesis of natural tyrosinases. Mechanistic investigations demonstrate that Cu-HC4 utilizes both Cu centers cooperatively for diphenol oxidation and requires O(2) for catalysis like natural Cu oxidases but follows a distinct catalytic pathway compared to those enzymes. Cryo-EM characterization of a tetrameric form of HC4 reveals slight deviations in the relative positions of the active site His residues that may account for differences in reactivity between Cu-HC4 and natural T3 Cu enzymes.