On the Permeation by Dioxygen of Urate Oxidase from Aspergillus flavus in Complex with Xanthine Anion: Dioxygen Pathways and a Portrait of the Enzyme Cavities from Molecular Dynamics Simulations in Water Solution

Abstract

This work describes molecular dynamics (MD) simulations in aqueous media for the complex of the homotetrameric urate oxidase (UOX) from Aspergillus flavus with xanthine anion (5) in the presence of dioxygen (O2 ). After 196.6 ns of trajectory from unrestrained MD, a O2 molecule was observed leaving the bulk solvent to penetrate the enzyme between two subunits, A/C. From here, the same O2 molecule was observed migrating, across subunit C, to the hydrophobic cavity that shares residue V227 with the active site. The latter was finally attained, after 378.3 ns of trajectory, with O2 at a bonding distance from 5. The reverse same O2 pathway, from 5 to the bulk solvent, was observed as preferred pathway under random acceleration MD (RAMD), where an external, randomly oriented force was acting on O2 . Both MD and RAMD simulations revealed several cavities populated by O2 during its migration from the bulk solvent to the active site or backwards. Paying attention to the last hydrophobic cavity that apparently serves as O2 reservoir for the active site, it was noticed that its volume undergoes ample fluctuations during the MD simulation, as expected from the thermal motion of a flexible protein, independently from the particular subunit and no matter whether the cavity is filled or not by O2 .

On Dioxygen and Substrate Access to Soluble Methane Monooxygenases: An all‐Atom Molecular Dynamics Investigation in Water Solution

“…This whole pattern indicates some preference by the O 2 molecule, albeit toward extremely broad gates, which can also serve for O 2 entrance . Therefore, the results of RAMD simulations contradict speculations that O 2 can have indiscriminate access to the active site from micropores in the four‐helix bundle…”

“…Our approach to ligand pathways between the bulk solvent and the active site of proteins has long been based on classical molecular dynamics (MD) in aqueous environment . Any such project with diiron proteins was long delayed in view of the heavy computational burden of parameterizing the diiron active site through the bonded model.…”

On Dioxygen Permeation of MaL Laccase from the Thermophilic Fungus Melanocarpus albomyces: An all‐Atom Molecular Dynamics Investigation

“…The bonded model would be confined to MaL laccase, if successful. Because of such a discouraging perspective, in the framework of our systematic investigation of small‐gas permeation of proteins, an alluring nonbonded dummy‐atom model for Cu(II) that includes the Jahn–Teller effect was developed . This model, developed for single Cu(II) proteins, could also be valid for the complex array of Cu(II) ions of laccases.…”

“…The unraveled pathway of O 2 expulsion from the enzyme can also be reasonably assumed to be a pathway for the molecule O 2 from the external solvent to the enzyme active site. To this concern, we have recently shown that the preferred pathway for O 2 expulsion under RAMD from the active site of urate oxidase coincides with the O 2 pathway from the bulk solvent to the enzyme active site under unbiased MD, affording reliability to the RAMD code.…”

“…Therefore, we took the challenge of simulating the pathway of O 2 through MaL laccase by computational techniques. We choose classical molecular dynamics (MD), as we had already done with other proteins and small gases, most recently with urate oxidase‐O 2 . MaL laccase presented a particularly challenging problem of the parameterization of a complex array of transition‐metal ions.…”