| Popis: |
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 O2 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 FeIII is positioned near Cys50, which consists of a ferredoxin domain with a 2.7 A distance 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. |
