Finding molecular dioxygen tunnels in homoprotocatechuate 2,3-dioxygenase: implications for different reactivity of identical subunits

Extradiol dioxygenases facilitate microbial aerobic degradation of catechol and its derivatives by activating molecular dioxygen and incorporating both oxygen atoms into their substrates. Experimental and theoretical studies have focused on the mechanism of the reaction at the active site. However, whether the catalytic rate is limited by O₂ access to the active site has not yet been explored. Here, we choose a recently solved X-ray structure of homoprotocatechuate 2,3-dioxygenase as a typical example to determine potential pathways for O₂ migration from the solvent into the enzyme center. On the basis of the trajectories of two 10-ns molecular dynamics simulations, implicit ligand sampling was used to calculate the 3D free energy map for O₂ inside the protein. The energetically optimal routes for O₂ diffusion were identified for each subunit of the homotetrameric protein structure. The O₂ tunnels formed because of thermal fluctuations were also characterized by connecting elongated cavities inside the protein. By superimposing the favorable O₂ tunnels on to the free energy map, both energetically and geometrically preferred O₂ pathways were determined, as also were the amino acids that may be critical for O₂ passage along these paths. Our results demonstrate that identical subunits possess quite distinct O₂ tunnels. The order of O₂ affinity of these tunnels is generally consistent with the order of the catalytic rate of each subunit. As a consequence, the probability of finding the reaction product is highest in the subunit containing the highest O₂ affinity pathway.