Methane monooxygenase(MMO)has attracted significant attention owing to its crucial role in the global carbon cycle;it impedes greenhouse effects by converting methane to methanol under ambient conditions.The water-sol...Methane monooxygenase(MMO)has attracted significant attention owing to its crucial role in the global carbon cycle;it impedes greenhouse effects by converting methane to methanol under ambient conditions.The water-soluble form of MMO(sMMO)has three essential components for the hydroxylation of methane:hydroxylase(MMOH),reductase(MMOR),and a regulatory(MMOB)component.MMOR consists of a flavin adenine dinucleotide(FAD)binding domain and a ferredoxin domain containing the[2Fe–2S]cluster for electron transfer from NADH to the di-iron sites in MMOH,which exerts its catalytic activity through O_(2)activation.Herein,the electronic structures of two cofactors,the FAD radical and[2Fe–2S]^(+),of reduced MMOR from Methylosinus sporium strain 5 were investigated.The results of multi-frequency and multi-technique electron paramagnetic resonance(EPR)spectroscopy of chemically reduced MMOR indicated the presence of a neutral flavin radical,and its detailed electronic structure was supported by density functional theory(DFT)calculations.The electronic and oxidation environments of[2Fe–2S]^(+)were further investigated using advanced EPR spectroscopy.Spectroscopic results confirmed that the oxidized state of Fe^(Ⅲ)is positioned near Cys50,which consists of a ferredoxin domain with a 2.7Ådistance between iron atoms.Our EPR spectroscopic results may provide a paradigm to elucidate the distribution of electronic densities of multiple cofactors in the enzyme,thus understanding its functional role.展开更多
基金supported by the National Research Foundation of Korea(NRF-2017M3D1A1039380 and NRF-2017R1A2B4008691)to S.H.K.S.J.Lthe Ministry of Education(2017R1A6A1A03015876)the C1 Gas Refinery Program(NRF-2015M3D3D3A1A01064876)for funding.
文摘Methane monooxygenase(MMO)has attracted significant attention owing to its crucial role in the global carbon cycle;it impedes greenhouse effects by converting methane to methanol under ambient conditions.The water-soluble form of MMO(sMMO)has three essential components for the hydroxylation of methane:hydroxylase(MMOH),reductase(MMOR),and a regulatory(MMOB)component.MMOR consists of a flavin adenine dinucleotide(FAD)binding domain and a ferredoxin domain containing the[2Fe–2S]cluster for electron transfer from NADH to the di-iron sites in MMOH,which exerts its catalytic activity through O_(2)activation.Herein,the electronic structures of two cofactors,the FAD radical and[2Fe–2S]^(+),of reduced MMOR from Methylosinus sporium strain 5 were investigated.The results of multi-frequency and multi-technique electron paramagnetic resonance(EPR)spectroscopy of chemically reduced MMOR indicated the presence of a neutral flavin radical,and its detailed electronic structure was supported by density functional theory(DFT)calculations.The electronic and oxidation environments of[2Fe–2S]^(+)were further investigated using advanced EPR spectroscopy.Spectroscopic results confirmed that the oxidized state of Fe^(Ⅲ)is positioned near Cys50,which consists of a ferredoxin domain with a 2.7Ådistance between iron atoms.Our EPR spectroscopic results may provide a paradigm to elucidate the distribution of electronic densities of multiple cofactors in the enzyme,thus understanding its functional role.
