Abstract
Methane, an important greenhouse gas, has a 20-fold higher heat capacity than carbon dioxide. Earlier, through advanced spectroscopy and structural studies, the mechanisms underlying the extremely stable C–H activation of soluble methane monooxygenase (sMMO) have been elucidated in Methylosinus trichosporium OB3b and Methylococcus capsulatus Bath. Here, sMMO components—including hydroxylase (MMOH), regulatory (MMOB), and reductase (MMOR)—were expressed and purified from a type II methanotroph, Methylosinus sporium strain 5 (M. sporium 5), to characterize its hydroxylation mechanism. Two molar equivalents of MMOB are necessary to achieve catalytic activities and oxidized a broad range of substrates including alkanes, alkenes, halogens, and aromatics. Optimal activities were observed at pH 7.5 for most substrates possibly because of the electron transfer environment in MMOR. Substitution of MMOB or MMOR from another type II methanotroph, Methylocystis species M, retained specific enzyme activities, demonstrating the successful cross-reactivity of M. sporium 5. These results will provide fundamental information for further enzymatic studies to elucidate sMMO mechanisms.
Highlights
Methanotrophic bacteria require methane gas (CH4 ) as the sole carbon and energy source [1,2,3]
For the expression of soluble methane monooxygenase (sMMO), M. sporium 5 was cultured in copper-limited nitrate mineral salt (NMS) media, and results
To elucidate the catalytic mechanisms of sMMO, M. sporium 5 can be used for isolating intermediates
Summary
Methanotrophic bacteria (methanotrophs) require methane gas (CH4 ) as the sole carbon and energy source [1,2,3]. Hydroxylation of methane by methanotrophs is a crucial process that regulates the carbon cycle in ecological systems and can be applied in the fields of bioremediation and bioenergy [4,5,6]. The level of CH4 in ecological systems is regulated by methane monooxygenases (MMOs). The membrane-bound methane monooxygenase (pMMO) is expressed in most methanotrophs, including type I, II, and X, and recent studies have demonstrated detailed structures of this enzyme and its active sites [9,10,11]. The other type of MMO, soluble methane monooxygenase (sMMO), has a non-heme diiron active site and is expressed in copper-limited conditions in limited type II and type X methanotrophs [1,4,6,12]
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