Structure and activity of particulate methane monooxygenase arrays in methanotrophs

Abstract

Methane-oxidizing bacteria play a central role in greenhouse gas mitigation and have potential applications in biomanufacturing. Their primary metabolic enzyme, particulate methane monooxygenase (pMMO), is housed in copper-induced intracytoplasmic membranes (ICMs), of which the function and biogenesis are not known. We show by serial cryo-focused ion beam (cryoFIB) milling/scanning electron microscope (SEM) volume imaging and lamellae-based cellular cryo-electron tomography (cryoET) that these ICMs are derived from the inner cell membrane. The pMMO trimer, resolved by cryoET and subtomogram averaging to 4.8 Å in the ICM, forms higher-order hexagonal arrays in intact cells. Array formation correlates with increased enzymatic activity, highlighting the importance of studying the enzyme in its native environment. These findings also demonstrate the power of cryoET to structurally characterize native membrane enzymes in the cellular context.

Unraveling the Valence State and Reactivity of Copper Centers in Membrane-Bound Particulate Methane Monooxygenase

“…19,37,45 However, recent investigations underscore the indispensability of the membrane environment for both pMMO's structure and function. 1,16,18 In the absence of the membrane environment, the purified pMMO exhibits lower activity compared to the native one with membrane environment. 16,26,46,47 In addition, the purification process resulted in the loss of approximately 25 residues neighboring the Cu C site within the PmoC subunit, implying the significance of the membrane environment in preserving the structural stability and integrity of pMMO.…”

“…[25][26][27]48 Intriguingly, the recent work unveiled a fourth copper binding site (labeled as Cu D , depicted in Figure 1) within lipid-nanodisc reconstituted pMMO, which is ligated with the conserved Asn227, His231, and His245 near the C-terminus of the PmoC subunit. 1,16 Notably, these three residues (Asn227, His231, and His245) located in the loop of PmoC are responsible for the Cu D binding, but they were missing in the purified pMMO. 2,19 Despite extensive experimental and computational investigations, the valence states and catalytic functionalities of both the Cu C and Cu D sites within membrane-bound pMMO remain elusive.…”

“…Methane monooxygenases (MMOs), pivotal in the conversion of methane to methanol, − play crucial roles in ecological carbon cycling and climate regulation. To date, two distinct varieties of MMOs have been elucidated.…”

“…To date, two distinct varieties of MMOs have been elucidated. One is the diiron-containing soluble methane monooxygenase (sMMO) from which an extensive array of structural and spectroscopic insights has been garnered. ,− The alternative family is the membrane-bound and copper-containing particulate methane monooxygenase (pMMO). − ,− Across all methanotrophs, pMMO expression is pervasive, enabling methane-utilizing bacteria to annually consume tens of millions of metric tons of atmospheric methane. ,,, …”

“…Apart from the Cu A and Cu B sites, an initial Zn-binding site within the PmoC subunit ,− ,, was subsequently revealed to be a copper-binding site (designated Cu C ), featuring Asp129, His133, and His146 as ligands (Figure ). ,,,,, This site is highly conserved and located in a disordered region of the PmoC subunit. , The Cu C site has been suggested as the catalytic active center within purified pMMO. ,, However, recent investigations underscore the indispensability of the membrane environment for both pMMO’s structure and function. ,, In the absence of the membrane environment, the purified pMMO exhibits lower activity compared to the native one with membrane environment. ,,, In addition, the purification process resulted in the loss of approximately 25 residues neighboring the Cu C site within the PmoC subunit, implying the significance of the membrane environment in preserving the structural stability and integrity of pMMO. , Indeed, the reconstitution of membrane-like lipid nanodiscs can effectively restore the native methane oxidation activity of pMMO. − , Intriguingly, the recent work unveiled a fourth copper binding site (labeled as Cu D , depicted in Figure ) within lipid-nanodisc reconstituted pMMO, which is ligated with the conserved Asn227, His231, and His245 near the C-terminus of the PmoC subunit. , Notably, these three residues (Asn227, His231, and His245) located in the loop of PmoC are responsible for the Cu D binding, but they were missing in the purified pMMO. , …”