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.　©　2024