The PmoB subunit of particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath): The CuI sponge and its function

Lu, Yu‐Jhang; Hung, Mu‐Cheng; Chang, Brian T.-A.; Lee, Tsu-Lin; Lin, Zhi-Han; Tsai, I.K.; Chen, Yaosheng; Chang, Chin-Shuo; Tsai, Yi‐Fang; Chen, Kelvin H.‐C.; Chan, Sunney I.; Yu, Steve S.‐F.

doi:10.1016/j.jinorgbio.2019.04.005

Cited by 18 publications

(46 citation statements)

References 45 publications

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“…However, the pMMO itself has proven challenging to fully characterise, and the nature and location of the site of O 2 activation and methane oxidation remains uncertain. To date, a diiron site located on the PmoC subunit (29), and multiple copper sites of different nuclearities located on separate subunits (PmoA, PmoB and PmoC) have all been suggested as potential active sites (27,(30)(31)(32)(33)(34).…”

Section: Downloaded Frommentioning

confidence: 99%

Inhibition of Ammonia Monooxygenase from Ammonia-Oxidizing Archaea by Linear and Aromatic Alkynes

Wright

Schatteman

Crombie

et al. 2020

Appl Environ Microbiol

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Ammonia monooxygenase (AMO) is a key nitrogen-transforming enzyme belonging to the same copper-dependent membrane monooxygenase family (CuMMO) as the particulate methane monooxygenase (pMMO). The AMO from ammonia-oxidizing archaea (AOA) is very divergent from both the AMO of ammonia-oxidizing bacteria (AOB) and the pMMO from methanotrophs, and little is known about the structure or substrate range of the archaeal AMO. This study compares inhibition by C2 to C8 linear 1-alkynes of AMO from two phylogenetically distinct strains of AOA, “Candidatus Nitrosocosmicus franklandus” C13 and “Candidatus Nitrosotalea sinensis” Nd2, with AMO from Nitrosomonas europaea and pMMO from Methylococcus capsulatus (Bath). An increased sensitivity of the archaeal AMO to short-chain-length alkynes (≤C5) appeared to be conserved across AOA lineages. Similarities in C2 to C8 alkyne inhibition profiles between AMO from AOA and pMMO from M. capsulatus suggested that the archaeal AMO has a narrower substrate range than N. europaea AMO. Inhibition of AMO from “Ca. Nitrosocosmicus franklandus” and N. europaea by the aromatic alkyne phenylacetylene was also investigated. Kinetic data revealed that the mechanisms by which phenylacetylene inhibits “Ca. Nitrosocosmicus franklandus” and N. europaea are different, indicating differences in the AMO active site between AOA and AOB. Phenylacetylene was found to be a specific and irreversible inhibitor of AMO from “Ca. Nitrosocosmicus franklandus,” and it does not compete with NH3 for binding at the active site. IMPORTANCE Archaeal and bacterial ammonia oxidizers (AOA and AOB, respectively) initiate nitrification by oxidizing ammonia to hydroxylamine, a reaction catalyzed by ammonia monooxygenase (AMO). AMO enzyme is difficult to purify in its active form, and its structure and biochemistry remain largely unexplored. The bacterial AMO and the closely related particulate methane monooxygenase (pMMO) have a broad range of hydrocarbon cooxidation substrates. This study provides insights into the AMO of previously unstudied archaeal genera, by comparing the response of the archaeal AMO, a bacterial AMO, and pMMO to inhibition by linear 1-alkynes and the aromatic alkyne, phenylacetylene. Reduced sensitivity to inhibition by larger alkynes suggests that the archaeal AMO has a narrower hydrocarbon substrate range than the bacterial AMO, as previously reported for other genera of AOA. Phenylacetylene inhibited the archaeal and bacterial AMOs at different thresholds and by different mechanisms of inhibition, highlighting structural differences between the two forms of monooxygenase.

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Section: Downloaded Frommentioning

confidence: 99%

Inhibition of Ammonia Monooxygenase from Ammonia-Oxidizing Archaea by Linear and Aromatic Alkynes

Wright

Schatteman

Crombie

et al. 2020

Appl Environ Microbiol

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“…Recently, those findings have been revised to conclude that pMMO is a protein with mononuclear copper ions at the A , B , and C sites only. , In contrast, there are 12–14 coppers found within each protomer of the functional pMMO according to spectroscopic and biochemical analyses from several other laboratories. − It appears that the majority of copper cofactors were inadvertently removed during preparation of the pMMO for protein crystallization. Because the copper cofactors constitute the catalytic machinery of the enzyme, these conflicting views, including the recent debates over the nuclearity of the copper center at the B site ,, and the location of the active site(s) ,,− have left a conundrum on understanding the mechanism of pMMO.…”

Section: Introductionmentioning

confidence: 99%

Copper Centers in the Cryo-EM Structure of Particulate Methane Monooxygenase Reveal the Catalytic Machinery of Methane Oxidation

Chang

et al. 2021

J. Am. Chem. Soc.

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The particulate methane monooxygenase (pMMO) is the first enzyme in the C1 metabolic pathway in methanotrophic bacteria. As this enzyme converts methane into methanol efficiently near room temperature, it has become the paradigm for developing an understanding of this difficult C1 chemistry. pMMO is a membranebound protein with three subunits (PmoB, PmoA, and PmoC) and 12− 14 coppers distributed among different sites. X-ray crystal structures that have revealed only three mononuclear coppers at three sites have neither disclosed the location of the active site nor the catalytic mechanism of the enzyme. Here we report a cyro-EM structure of holo-pMMO from Methylococcus capsulatus (Bath) at 2.5 Å, and develop quantitative electrostatic-potential profiling to scrutinize the nonprotein densities for signatures of the copper cofactors. Our results confirm a mononuclear Cu I at the A site, resolve two Cu I s at the B site, and uncover additional Cu I clusters at the PmoA/PmoC interface within the membrane (D site) and in the water-exposed C-terminal subdomain of the PmoB (E clusters). These findings complete the minimal set of copper factors required for catalytic turnover of pMMO, offering a glimpse of the catalytic machinery for methane oxidation according to the chemical principles underlying the mechanism proposed earlier.

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“…The study found that all four characteristic peaks changed during the preparation of Mb–Cu when Cu(II) was added to the Mb solution. When the main peak no longer changed, it was considered that the coordination of Mb and copper was saturated [31].…”

Section: Resultsmentioning

confidence: 99%

“…These unrelated distinct functions of Mb vary with changes in its cell location, physical environment, and complex formation with pMMO, suggesting that Mb may belong to a group of proteins known as moonlighting proteins [29,30]. It was reported that the PmoB subunit is a Cu(I) protein with many copper-binding sites [31]. There is a mononuclear copper site and a dicopper site in the N-terminal sub-domain and “E-clusters” associated with the C-terminal sub-domain of the PmoB subunit [31].…”

Section: Introductionmentioning

confidence: 99%

“…It was reported that the PmoB subunit is a Cu(I) protein with many copper-binding sites [31]. There is a mononuclear copper site and a dicopper site in the N-terminal sub-domain and “E-clusters” associated with the C-terminal sub-domain of the PmoB subunit [31]. The “E-clusters” provide a reservoir of reducing equivalents to re-reduce the tricopper cluster of the proposed active site after the latter cofactor completes the oxidative phase of the enzyme turnover [31].…”

Section: Introductionmentioning

confidence: 99%

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Hybridization of Particulate Methane Monooxygenase by Methanobactin-Modified AuNPs

et al. 2019

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Particulate methane monooxygenase (pMMO) is a characteristic membrane-bound metalloenzyme of methane-oxidizing bacteria that can catalyze the bioconversion of methane to methanol. However, in order to achieve pMMO-based continuous methane-to-methanol bioconversion, the problems of reducing power in vitro regeneration and pMMO stability need to be overcome. Methanobactin (Mb) is a small copper-chelating molecule that functions not only as electron carrier for pMMO catalysis and pMMO protector against oxygen radicals, but also as an agent for copper acquisition and uptake. In order to improve the activity and stability of pMMO, methanobactin–Cu (Mb–Cu)-modified gold nanoparticle (AuNP)–pMMO nanobiohybrids were straightforwardly synthesized via in situ reduction of HAuCl4 to AuNPs in a membrane fraction before further association with Mb–Cu. Mb–Cu modification can greatly improve the activity and stability of pMMO in the AuNP–pMMO nanobiohybrids. It is shown that the Mb–Cu-modified AuNP–pMMO nanobiohybrids can persistently catalyze the conversion of methane to methanol with hydroquinone as electron donor. The artificial heterogeneous nanobiohybrids exhibited excellent reusability and reproducibility in three cycles of catalysis, and they provide a model for achieving hydroquinone-driven conversion of methane to methanol.

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The PmoB subunit of particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath): The CuI sponge and its function

Cited by 18 publications

References 45 publications

Inhibition of Ammonia Monooxygenase from Ammonia-Oxidizing Archaea by Linear and Aromatic Alkynes

Inhibition of Ammonia Monooxygenase from Ammonia-Oxidizing Archaea by Linear and Aromatic Alkynes

Copper Centers in the Cryo-EM Structure of Particulate Methane Monooxygenase Reveal the Catalytic Machinery of Methane Oxidation

Hybridization of Particulate Methane Monooxygenase by Methanobactin-Modified AuNPs

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