Pulsed EPR Studies of Particulate Methane Monooxygenase from <i>Methylococcus Capsulatus</i> (Bath):  Evidence for Histidine Ligation

Elliott, Sean J.; Randall, David; Britt, and R. David; Chan, Sunney I.

doi:10.1021/ja972968+

Cited by 41 publications

(55 citation statements)

References 25 publications

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“…PmoC and PmoA are highly hydrophobic proteins with six predicted transmembrane-spanning regions, whereas PmoB is probably inserted into the membrane with only two helices. The predicted polypeptides contain 17 histidine residues in total for M. trichosporium (8). Likewise, there are 4 conserved histidine residues each in PmoB and AmoB and in PmoC and AmoC, and although these polypeptides probably do not contain the active site of the enzyme, it is still possible that they provide ligands for copper ions at the active site.…”

Section: Discussionmentioning

confidence: 99%

Molecular Analysis of the pmo (Particulate Methane Monooxygenase) Operons from Two Type II Methanotrophs

Gilbert

McDonald

Finch

et al. 2000

Appl Environ Microbiol

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The particulate methane monooxygenase gene clusters, pmoCAB, from two representative type II methanotrophs of the ␣-Proteobacteria, Methylosinus trichosporium OB3b and Methylocystis sp. strain M, have been cloned and sequenced. Primer extension experiments revealed that the pmo cluster is probably transcribed from a single transcriptional start site located 300 bp upstream of the start of the first gene, pmoC, for Methylocystis sp. strain M. Immediately upstream of the putative start site, consensus sequences for 70 promoters were identified, suggesting that these pmo genes are recognized by 70 and negatively regulated under low-copper conditions. The pmo genes were cloned in several overlapping fragments, since parts of these genes appeared to be toxic to the Escherichia coli host. Methanotrophs contain two virtually identical copies of pmo genes, and it was necessary to use Southern blotting and probing with pmo gene fragments in order to differentiate between the two pmoCAB clusters in both methanotrophs. The complete DNA sequence of one copy of pmo genes from each organism is reported here. The gene sequences are 84% similar to each other and 75% similar to that of a type I methanotroph of the ␥-Proteobacteria, Methylococcus capsulatus Bath. The derived proteins PmoC and PmoA are predicted to be highly hydrophobic and consist mainly of transmembranespanning regions, whereas PmoB has only two putative transmembrane-spanning helices. Hybridization experiments showed that there are two copies of pmoC in both M. trichosporium OB3b and Methylocystis sp. strain M, and not three copies as found in M. capsulatus Bath.Methane-oxidizing bacteria (methanotrophs) play an important part in the global carbon cycle, recycling up to 60% (680 Tg) of total global methane production per year (25). Methane is used as the sole source of carbon and energy by these organisms. It is oxidized to methanol by the key enzyme methane monooxygenase (MMO). Methanol is further oxidized to formaldehyde. Formaldehyde is then either assimilated into cell biomass or oxidized via formate to carbon dioxide. All known methanotrophs possess the membrane-bound or particulate form of MMO (pMMO), and some have a second enzyme, the cytoplasmic, or soluble, MMO (sMMO).Two types of methanotrophs can be distinguished on the basis of biochemical and ultrastructural differences (3, 33). Genetic and biochemical work has been carried out mainly on two organisms, the type I methanotroph Methylococcus capsulatus Bath, a ␥-proteobacterium, and the type II methanotroph Methylosinus trichosporium OB3b, an ␣-proteobacterium. Another well-studied type II organism, Methylocystis sp. strain M, was isolated from a trichloroethylene-degrading mixed culture (20, 32). The sMMOs of these bacteria are very similar (5, 10, 20), and their sMMO gene sequences are highly conserved (17).Recently, the pMMO was purified from M. capsulatus Bath (21, 34) and M. trichosporium OB3b (31). It is a copper-containing monooxygenase which is oxygen and light sensitive. The 26-kDa subunit o...

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Section: Discussionmentioning

confidence: 99%

Molecular Analysis of the pmo (Particulate Methane Monooxygenase) Operons from Two Type II Methanotrophs

Gilbert

McDonald

Finch

et al. 2000

Appl Environ Microbiol

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“…15 N-labeling of the enzyme confirmed that the superhyperfine structure is derived from nuclear coupling to nitrogens. From ESEEM and ENDOR experiments, 21 we have previously concluded that these nitrogens are from the imidazoles of histidines. Three and four nitrogen-bearing ligands have been suggested in the literature; 15 …”

Section: Ligand Structure Of the Type 2 Centermentioning

confidence: 99%

“…Apparently, no 14 N-superhyperfine structures were associated with the EPR of the E-cluster copper ions when they became oxidized, as was previously concluded from ESEEM studies. 21 The amount of dithionite required to obtain the protein sample with the "null" EPR signal was used to scale up the production of the fully reduced pMMO. Aliquots of the fully reduced protein were then prepared, and the pMMO samples were re-oxidized by adding varying amounts of a 0.1% hydrogen peroxide solution.…”

Section: Scheme Imentioning

confidence: 99%

“…20 Further studies have indicated that there must be at least two histidine residues at the active site of pMMO (C-clusters) by pulsed EPR and electron spin echo envelope modulation (ESEEM) experiments. 21 The exact nature of the copper centers in the pMMO is very complex. Their geometric arrangements, ligand structures and their function during catalysis are still unclear.…”

Section: Introductionmentioning

confidence: 99%

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The Copper Clusters in the Particulate Methane Monooxygenase (pMMO) from Methylococcus Capsulatus (Bath)

Chen

Tseng

et al. 2004

J Chinese Chemical Soc

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The particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) is a multicopper protein that hydroxylates methane and other small n-alkanes to their corresponding alcohols with high regiospecificity and stereoselectivity. The copper ions appear to be arranged into~5 trinuclear copper clusters. Two of these clusters are thought to be involved in the dioxygen chemistry and alkane hydroxylation mediated by the enzyme. Accordingly, these catalytic clusters (C-clusters) are typically oxidized, as the protein is isolated. The remaining copper ions are normally reduced, and it has been suggested that they provide a reservoir of reducing equivalents needed for the turnover of the enzyme. In this study, we have oxidized the protein to different levels of oxidation of the copper ions using dioxygen, hydrogen peroxide, ferricyanide, and dioxygen in the presence of the suicide substrate acetylene, and have characterized the oxidized copper centers using low temperature electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy. The results are consistent with the grouping of the copper ions into catalytic clusters and electron transfer clusters (E-clusters). Quantification indicates that there are, indeed, two C-clusters. A model in which the two C-clusters are activated by dioxygen starting from their fully reduced states and mediate the hydroxylation chemistry of methane will be presented and discussed.

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“…In this regard polynuclear copper complexes have attracted a special attention with respect to the biological role played by them. The driving force for further investigation of complexes containing three copper atoms lies in the recognition that the perticulate methane monooxygenase (pMMO) from Methylococcus capsulates (both) is constituted of 15 copper atom active center which is further organized into two types of five trinuclear aggregates of yet unknown structure [38,39]. The molecule counts in this pMMO are 12.8 copper and 0.9 iron atoms.…”

Section: Introductionmentioning

confidence: 99%

Synthesis, crystal structure, magnetic property and oxidative DNA cleavage activity of an octanuclear copper(II) complex showing water–perchlorate helical network

Dhara

Ratha

Manassero

et al. 2007

Journal of Inorganic Biochemistry

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Pulsed EPR Studies of Particulate Methane Monooxygenase from Methylococcus Capsulatus (Bath): Evidence for Histidine Ligation

Cited by 41 publications

References 25 publications

Molecular Analysis of the pmo (Particulate Methane Monooxygenase) Operons from Two Type II Methanotrophs

Molecular Analysis of the pmo (Particulate Methane Monooxygenase) Operons from Two Type II Methanotrophs

The Copper Clusters in the Particulate Methane Monooxygenase (pMMO) from Methylococcus Capsulatus (Bath)

Synthesis, crystal structure, magnetic property and oxidative DNA cleavage activity of an octanuclear copper(II) complex showing water–perchlorate helical network

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