Probing the molecular details of particulate methane monooxygenase in micelles to cells

Ro, Soo Yeon

doi:https://doi.org/10.21985/n2-knsa-h648

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Probing the molecular details of particulate methane monooxygenase in micelles to cells

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Harnessing the metabolic potential of methanotrophic bacteria is a compelling strategy for the bioremediation of environmentally harmful methane gas. Methanotrophs can activate a 105 kcal/mol C-H bond in methane at ambient conditions using metalloenzymes called methane monooxygenases (MMOs). Particulate methane monooxygenase (pMMO) is a copper-dependent, membrane-bound enzyme that is the predominant biological methane sink in nature. Despite its significant impact on the global carbon cycle, a molecular and mechanistic understanding of the pMMO active site is limited. The challenges stem from the structural complexity of pMMO and difficulties in manipulating the host organisms. At the beginning of this dissertation work in 2014, multiple crystal structures had already been obtained, which showed a protomer comprised of three subunits, PmoB, PmoA, and PmoC, assembled into a larger trimeric complex. These structures, along with spectroscopic studies, identified three metal centers that may house the catalytic copper ions, the CuB, bis-His, and CuC sites. However, low resolution structures, a mixture of copper species, and low enzymatic activity hampered characterization of the active site. Furthermore, the lack of a heterologous expression system or facile mutagenesis of methanotrophs limited pMMO studies to traditional biochemical and spectroscopic methods. Hence in this thesis dissertation, pMMO was investigated in native-like environments to more closely mimic its in vivo structure and function. pMMO reconstituted into membrane-mimetics exhibited methane oxidation activity, which confirmed the importance of studying pMMO in a membrane-dependent context. The active enzyme-membrane complex was further characterized via native top-down mass spectrometry (nTDMS) metal localization studies, which provided evidence that a monocopper CuC site was essential for activity. Recently developed genetic toolkits were applied to pMMO mutagenesis in an attempt to elucidate the essential residues involved in activity. Furthermore, the scope of this thesis was extended beyond pMMO to include isolation of a lanthanide-dependent methanol dehydrogenase and study of its interaction with pMMO. Additionally, mutagenesis of the extended pmo and mbn operons helped to identify key enzymes involved in copper uptake and transport. This dissertation highlights the benefits of interdisciplinary approaches that will shape future pMMO investigations.

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