Effect of methanobactin on the activity and electron paramagnetic resonance spectra of the membrane-associated methane monooxygenase in Methylococcus capsulatus Bath

Abstract

Improvements in the purification of methanobactin (mb) from either Methylosinus trichosporium OB3bT or Methylococcus capsulatus Bath resulted in preparations that stimulated methane-oxidation activity in both whole-cell and cell-free fractions of Methylococcus capsulatus Bath expressing the membrane-associated methane monooxygenase (pMMO). By using washed membrane factions with pMMO activities in the 290 nmol propylene oxidized min "1 (mg protein) "1 range, activities approaching 400 nmol propylene oxidized min "1 (mg protein) "1 were commonly observed following addition of copper-containing mb (Cu-mb), which represented 50-75 % of the total whole-cell activity.The stimulation of methane-oxidation activity by Cu-mb was similar to or greater than that observed with equimolar concentrations of Cu(II), without the inhibitory effects observed with high copper concentrations. Stimulation of pMMO activity was not observed with copper-free mb, nor was it observed when the copper-to-mb ratio was <0?5 Cu atoms per mb. The electron paramagnetic resonance (EPR) spectra of mb differed depending on the copper-to-mb ratio. At copper-to-mb ratios of <0?4 Cu(II) per mb, Cu(II) addition to mb showed an initial coordination by both sulfur and nitrogen, followed by reduction to Cu(I) in <2 min. At Cu(II)-to-mb ratios between 0?4 and 0?9 Cu(II) per mb, the intensity of the Cu(II) signal in EPR spectra was more representative of the Cu(II) added and indicated more nitrogen coordination. The EPR spectral properties of mb and pMMO were also examined in the washed membrane fraction following the addition of Cu(II), mb and Cu-mb in the presence or absence of reductants (NADH or duroquinol) and substrates (CH 4 and/or O 2 ). The results indicated that Cu-mb increased electron flow to the pMMO, increased the free radical formed following the addition of O 2 and decreased the residual free radical following the addition of O 2 plus CH 4 . The increase in pMMO activity and EPR spectral changes to the pMMO following Cu-mb addition represent the first positive evidence of interactions between the pMMO and Cu-mb.

Methane monooxygenase gene expression mediated by methanobactin in the presence of mineral copper sources

“…These compounds, originally called Cu-binding cofactor/compounds (cbc) (20,21) or Cu-binding ligands (CBL) (22), were initially assumed to act as Cu transporters (maybe chaperones) that somehow inserted Cu into pMMO for function. However, evidence has since shown that mb (aka cbc or CBL) is not likely intrinsically associated with pMMO (13), although Choi et al (10) have shown that the Cu-mb complex increases pMMO activity in cell-free and whole-cell preparations (relative to Cu or mb alone). Despite this observation, an environmental role for mb that is pertinent to these organisms in their natural habitat has not been established.…”

“…We propose that in situ methanotrophic ecology is controlled by the relative ability of some methanotrophs to mobilize and acquire Cu from mineral and organic solid phases to support pMMO expression. Specifically, a small, fluorescent chromopeptide, called methanobactin (mb) (9)(10)(11), was purified from Methylosinus trichosporium OB3b (a type II methanotroph that expresses both pMMO and sMMO), which mediates Cu acquisition and promotes pMMO expression in this organism. We suggest that Cu sequestration by mb from environmental Cu sources is the rate-limiting step in in situ pMMO expression, and, as such, mb:Cu interactions can explain where and when pMMO is expressed, methanotroph ecology, and possibly methane oxidation patterns in nature.…”

Variations in methanobactin structure influences copper utilization by methane-oxidizing bacteria

“…The mbtin (two forms) from Methylosinus trichosporium OB3b (a switchover organism) is the most extensively studied (13,(15)(16)(17)(25)(26)(27)(28)(29), and binds a single copper ion coordinated in a distorted tetrahedral arrangement by the nitrogens from two oxazolone rings (29) and the sulfurs from two enethiolate groups. The molecule has a compact arrangement stabilized by a disulfide bridge.…”

Biobased Synthesis of Gold Nanoparticles by Methanobactin

“…Mb from the spent medium of Methylosinus trichosporium 3011 was isolated as previously described for Methylococcus capsulatus Bath by Choi et al [6] and Methylosinus trichosporium OB3b by Kim et al [10] . The cells were removed by centrifugation at 10,000× g for 30 min.…”

“…Methanobactin (Mb) is a copper-binding small peptide that appears to function as an agent for copper detoxification, sequestration and uptake in methanotrophs. Mb has been identified in the extracellular fractions of both Methylosinus trichosporium OB3b [4,5] and Methylococcus capsulatus Bath [6] . Mb is composed of a tetrapeptide, a tripeptide, and several unusual moieties.…”