Statistical coupling analysis uncovers sites crucial for the proton transfer in laccase Lac15.

Laccases (benzenediol: oxygen oxidoreductases, EC1.10.3.2) can oxidize wide range of compounds thus have great application potential in diverse industries. The catalytic mechanisms of laccases have been extensively studied, while the details of proton transfer remain to be fully elucidated. In this study, we tried to uncover the sites that are crucial for the proton transfer of microbial laccase Lac15. A residue near the trinuclear copper center, D396, was indicated by statistical coupling analysis (SCA) and structural alignment to be an important site like D93, which is conserved in laccases and believed crucial for the catalysis by facilitating proton transfer. A representative mutant at this site, D396A, similar to D93A, exhibited significantly impaired catalysis with the global structure and substrate binding slightly perturbed. The mutation resulted in stay of the intermediate I, which would accept a proton to proceed to next catalysis stage, suggesting D396 might play a critical role in the proton transfer. Our finding may help to completely elucidate the proton transfer mechanism in laccases.