Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath)

Abstract

An active preparation of the membrane-associated methane monooxygenase (pMMO) from Methylococcus capsulatus Bath was isolated by ion-exchange and hydrophobic interaction chromatography using dodecyl ␤-D-maltoside as the detergent. The active preparation consisted of three major polypeptides with molecular masses of 47,000, 27,000, and 25,000 Da. Two of the three polypeptides (those with molecular masses of 47,000 and 27,000 Da) were identified as the polypeptides induced when cells expressing the soluble MMO are switched to culture medium in which the pMMO is expressed. The 27,000-Da polypeptide was identified as the acetylene-binding protein. The active enzyme complex contained 2.5 iron atoms and 14.5 copper atoms per 99,000 Da. The electron paramagnetic resonance spectrum of the enzyme showed evidence for a type 2 copper center (g-‫؍‬ 2.057, gሻ ‫؍‬ 2.24, and ͦAሻͦ ‫؍‬ 172 G), a weak high-spin iron signal (g ‫؍‬ 6.0), and a broad low-field (g ‫؍‬ 12.5) signal. Treatment of the pMMO with nitric oxide produced the ferrous-nitric oxide derivative observed in the membrane fraction of cells expressing the pMMO. When duroquinol was used as a reductant, the specific activity of the purified enzyme was 11.1 nmol of propylene oxidized ⅐ min ؊1 ⅐ mg of protein ؊1, which accounted for approximately 30% of the cell-free propylene oxidation activity. The activity was stimulated by ferric and cupric metal ions in addition to the cytochrome b-specific inhibitors myxothiazol and 2-heptyl-4-hydroxyquinoline-N-oxide.In methanotrophs, the oxidation of methane to methanol is catalyzed by the methane monooxygenase (MMO) (31). In some genera, either a soluble or a membrane-associated MMO is present depending on the copper concentration during growth (13,43,50,54). At low copper-to-biomass ratios, the enzyme activity occurs in the soluble fraction and is referred to as the soluble MMO (sMMO). At higher copper-to-biomass ratios, methane oxidation activity is catalyzed in the membrane fraction by the membrane-associated or particulate MMO (pMMO). The polypeptides and genes for the sMMO in several different methanotrophs have been characterized (10, 18, 19, 22-24, 34, 41, 42, 54). The pMMO has proven more elusive. There is indirect evidence that the enzyme contains copper, and inhibitor studies indicate that the enzyme is coupled to the electron transport chain (13,40,52,54). In addition, three polypeptides with molecular masses of 46,000, 35,000, and 26,000 Da are induced when cells expressing the sMMO convert to expression of pMMO under altered growth conditions (13, 54). Tonge et al. (55) reported the solubilization of methane-oxidizing activity from Methylosinus trichosporium OB3b with phospholipase C or by sonification. A three-subunit enzyme with subunit molecular masses of 47,000, 13,000, and 9,400 Da was isolated from this solubilized fraction. The 13,000-Da polypeptide was a CO-binding c-type cytochrome. The 47,000-Da polypeptide contained approximately one copper atom per molecule, and the 9,400-Da polypeptide was reported to...

Inactivation of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath) by acetylene

“…Earlier workers have used [ 14 C]-labeled HCCH as the mechanism probe of the catalytic site and have detected the radioactivity primarily in the "~27 kDa" subunit in denaturing SDS-PAGE gels of the protein [8,18]. In the latter work [8], the labeling experiment is not clean, leading to the observation of [ 14 C]-radioactivity in the protein band corresponding to the PmoB subunit as well as other cytoplasmic proteins ( Figure 3 of ref 18) Subsequently, the "~27 kDa" subunit was assigned to PmoA by N-terminal sequencing [8].…”

“…For this reason, HCCH is considered to be a mechanism-based probe of the enzyme catalytic site. Earlier experiments on cell extracts [18], whole cells [8], and the purified pMMO [8] using [ 14 C]-labeled HCCH as the substrate have shown that only the "~27 kDa" protein band on SDS-PAGE gels [18] is labeled with radioactivity, and N-terminal sequencing was used to assign this protein band to the PmoA subunit [8]. This observation suggests that the catalytic site of the enzyme resides within the…”

“…However, it is well known that the activities of pMMO in preparations vary in the order cells > purified membrane > reconstituted protein in the detergent micelles. Moreover, earlier studies on the inhibition of pMMO by [ 14 C]-acetylene conducted on cell extracts [18], whole cells [8] or purified pMMO with good specific activity [8] gave the same labeling pattern of pMMO. For inactivation of an enzyme by a mechanism-based inhibitor, it is not possible to derive any information unless substantial portions of the proteins are active to begin with.…”

“…Earlier suicide substrate experiments using radioactive HCCH to label the subunits, followed by SDS-PAGE, and N-terminal amino acid sequencing [8] have implicated PmoA (earlier (vide infra) assignment of the "~27 kDa" protein band in the SDS-PAGE gel) as the subunit labeled. Thus, our identification of PmoC as the subunit modified by HCCH is at odds with the earlier conclusion.…”

“…It is a large protein complex with three subunits, PmoA, PmoB and PmoC, and many copper ions [1,5]. Highly active protein preparations consisting of the full complement of 12-15 copper ions per protein monomer have been reported [6][7][8][9]. However, crystal structure studies from different species [10][11][12] have appeared showing the trimeric architecture of the pMMO complexes, each consisting of one copy of the PmoA, PmoC, and PmoC subunits, but with just 2-3 coppers and one zinc ion per monomer.…”