Locally enhanced sampling study of dioxygen diffusion pathways in homoprotocatechuate 2,3-dioxygenase

Extradiol dioxygenases are characterized by activating dioxygen and incorporating both oxygen atoms into their substrates. Both experimental and theoretical investigations have been focused on the detection of intermediates in the reaction cycle in order to develop a general chemical mechanism of O(2) activation and insertion. However, little is known about the mechanism of how O(2) reaches the reaction sites of the related enzymes, which raises the question whether the rate of catalysis is limited by O(2) access to the active site. In this paper, two locally enhanced sampling molecular dynamics simulations were performed to determine the potential O(2) pathways inside a recently solved X-ray structure of homoprotocatechuate 2,3-dioxygenase. It is found that nominally identical subunits of the single homotetrameric structure contain distinct O(2) affinity diffusion pathways, which partly correlates with the observation of the simultaneous presence of three different reaction intermediates in four independent active sites. Residues that are critical for O(2) diffusion are also examined and discussed. In particular, we find that the breathing motion of the internal cavity defined by these residues results in O(2) migration process.