The hydroxylation of methane (CH4) is crucial to the field of environmental microbiology, owing to the heat capacity of methane, which is much higher than that of carbon dioxide (CO2). Soluble methane monooxygenase (sMMO), a member of the bacterial multicomponent monooxygenase (BMM) superfamily, is essential for the hydroxylation of specific substrates, including hydroxylase (MMOH), regulatory component (MMOB), and reductase (MMOR). The diiron active site positioned in the MMOH α-subunit is reduced through the interaction of MMOR in the catalytic cycle. The electron transfer pathway, however, is not yet fully understood due to the absence of complex structures with reductases. A type II methanotroph, Methylosinus sporium 5, successfully expressed sMMO and hydroxylase, which were purified for the study of the mechanisms. Studies on the MMOH-MMOB interaction have demonstrated that Tyr76 and Trp78 induce hydrophobic interactions through π-π stacking. Structural analysis and sequencing of the ferredoxin domain in MMOR (MMOR-Fd) suggested that Tyr93 and Tyr95 could be key residues for electron transfer. Mutational studies of these residues have shown that the concentrations of flavin adenine dinucleotide (FAD) and iron ions are changed. The measurements of dissociation constants (Kds) between hydroxylase and mutated reductases confirmed that the binding affinities were not significantly changed, although the specific enzyme activities were significantly reduced by MMOR-Y93A. This result shows that Tyr93 could be a crucial residue for the electron transfer route at the interface between hydroxylase and reductase.
Keywords: Soluble methane monooxygenase (sMMO); bacterial multicomponent monooxygenase (BMM); electron transfer; hydroxylation; reductase.