Structural Studies of Particulate Methane Monooxygenase in a Native Lipid Bilayer

Koo, Christopher Wellington

doi:https://doi.org/10.21985/n2-kk3p-vb51

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Structural Studies of Particulate Methane Monooxygenase in a Native Lipid Bilayer

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Methanotrophs, bacteria that can metabolize methane, remain a promising solution to mitigating the effects of climate change by removing atmospheric methane and converting it to useful chemical precursors. However, a full understanding of the main enzyme they use to oxidize methane, particulate methane monooxygenase (pMMO), is critical for harnessing their unique metabolism. pMMO has several features that have made it a challenging enzyme to study. pMMO is a multi-subunit membrane enzyme, precluding a recombinant expression system and requiring disruptive detergents to isolate from the cell membrane, causing a loss of activity with each step of purification. It requires copper for activity and yet too much copper is inhibitory. In addition, advanced spectroscopy remains difficult since only Cu(II) has an electron paramagnetic resonance (EPR) signature while Cu(I) is EPR-silent. Finally, while genetic tools for methanotrophs are available, pMMO continues to resist mutagenesis studies. Despite these challenges, steady progress has been made on understanding the enzyme by applying cutting-edge techniques and taking new approaches. At the beginning of this dissertation work in 2016, crystal structures from multiple species of methanotrophs consistently revealed the trimer-of-trimers structure composed of subunits PmoA, PmoB, and PmoC, and two conserved copper binding sites, CuB and CuC. During this time, attention had started to shift from the periplasmic CuB site to the strictly conserved CuC site embedded in the membrane as the possible active site. CuC was shown to be labile compared to CuB and could be replaced with zinc, inhibiting activity. Concerningly, there was always ~25 amino acid stretch of disorder in the crystal structures near CuC that contained strictly conserved residues. Additionally, CuB could be modeled as both mono- or dinuclear copper ions in some pMMO crystal structures. During the course of this work, efforts focused on re-creating the membrane environment around pMMO in order to study it in a native-like environment. Bicelles successfully restored enzymatic activity and nanodiscs were shown to recover activity as well. At the same time, a double electron-electron resonance (DEER) study showed that CuC in purified pMMO samples contained copper and electron-nuclear double resonance (ENDOR) was used to determine that CuB was mononuclear. A native top-down mass spectrometry study on pMMO embedded in nanodiscs linked enzyme activity with the presence of at least one copper in thePmoC subunit. In this thesis dissertation, three more techniques were used to characterize pMMO: single-particle cryogenic electron microscopy (cryoEM), cryogenic electron tomography (cryoET), and cell-free protein synthesis (CFPS). The cryoEM study resulted in the highest resolution structure (2.14 Å) of pMMO to date in a nanodisc with lipids derived from the methanotroph and structures from three species in total. These structures uncovered stabilized lipids within the enzyme, structural features in the previously disordered region, and a new copper site (CuD), creating a new paradigm for modeling the architecture of the active site. A collaborative study using cryoET resulted in the highest resolution structure of a membrane protein using this technique (4.8 Å) and revealed the in vivo array organization of pMMO within the specialized intracytoplasmic membranes that stretch across the body of the methanotroph. Finally, the CFPS study resulted in the foundation of a viable recombinant platform for expressing the pMMO complex and generating mutants. Together, these studies highlight the importance of investigating pMMO in its native environment and highlights new possibilites that cryoEM and synthetic biology techniques can bring to future pMMO studies.

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