The Pathway for O₂ Diffusion inside CotA Laccase and Possible Implications on the Multicopper Oxidases Family

Laccases and multicopper oxidases (MCOs) oxidize a wide range of organic compounds while reducing O₂ to water, enabling numerous biotechnological applications. It is still unknown how O₂ reaches the internalized catalytic center of MCOs where it gets reduced, despite a proposed channel inferred from X-ray crystallography structures. Herein, an alternative new pathway is found through the use of a combination of free energy calculations (implicit ligand sampling), landscape analysis, and Markov modeling. The reported pathway is shown to be the one mostly contributing to O₂ reaching the catalytic center. This pathway is considered in light of the whole MCO family, and a relation to the protonation state of a structurally conserved acidic residue right above the center is advanced.