Theoretical insights into the formation of oxygen activation sites with the affection of protein microenvironment in Flavin-Dependent monooxygenases MsuD

[Display omitted] •A reasonable oxygen binding structure which responses to the reaction N5 site in MsuD enzyme is identified.•An oxygen migration channel in MsuD enzyme is revealed to support the formation of the identified oxygen binding structure.•Residue Asn104 plays an important role for maintaining this oxygen binding structure in the reaction space of MsuD enzyme.•Under this O2 binding structure, the N5 site is prioritized over C4a as the reaction site. Flavin-dependent monooxygenases are very famous for their capability to catalyze the oxidation of various substrates using oxygen and reduced flavin mononucleotide (FMNH-) or reduced flavin adenine dinucleotide (FADH-). When investigating the catalytic mechanism of flavin-dependent monooxygenase systems, a fundamental problem remains unsolved is that the O2 binding scheme within the reaction cavity is unclear. By applying molecular dynamics simulations, MDpocket simulations, residue mutation molecular dynamics simulation and energy decomposition analyses, this work identified the O2 binding pockets and discovered a possible oxygen migration channel within MsuD enzyme. Molecular dynamics simulations after residue mutation proved that residues Asn104 plays an important role in maintaining the O2 position within the reaction cavity. By analyzing the interaction between O2 and the surrounding protein microenvironment, we demonstrated that N5 is a more favored reaction site and speculated a reasonable O2 activation process. This work recovers the formation of oxygen activation sites in MsuD enzyme, which is of great significance to further investigations of the catalytic reaction mechanism. This work provides a feasible method to study the O2 binding in proteins and the role of the protein microenvironment around the reaction cavity.