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, 360 (1458), 1207-22

The Leeuwenhoek Lecture 2000 the Natural and Unnatural History of Methane-Oxidizing Bacteria

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The Leeuwenhoek Lecture 2000 the Natural and Unnatural History of Methane-Oxidizing Bacteria

Howard Dalton. Philos Trans R Soc Lond B Biol Sci.

Abstract

Methane gas is produced from many natural and anthropogenic sources. As such, methane gas plays a significant role in the Earth's climate, being 25 times more effective as a greenhouse gas than carbon dioxide. As with nearly all other naturally produced organic molecules on Earth, there are also micro-organisms capable of using methane as their sole source of carbon and energy. The microbes responsible (methanotrophs) are ubiquitous and, for the most part, aerobic. Although anaerobic methanotrophs are believed to exist, so far, none have been isolated in pure culture. Methanotrophs have been known to exist for over 100 years; however, it is only in the last 30 years that we have begun to understand their physiology and biochemistry. Their unique ability to use methane for growth is attributed to the presence of a multicomponent enzyme system-methane monooxygenase (MMO)-which has two distinct forms: soluble (sMMO) and membrane-associated (pMMO); however, both convert methane into the readily assimilable product, methanol. Our understanding of how bacteria are capable of effecting one of the most difficult reactions in chemistry-namely, the controlled oxidation of methane to methanol-has been made possible by the isolation, in pure form, of the enzyme components.The mechanism by which methane is activated by sMMO involves abstraction of a hydrogen atom from methane by a high-valence iron species (FeIV or possibly FeV) in the hydroxylase component of the MMO complex to form a methyl radical. The radical combines with a captive oxygen atom from dioxygen to form the reaction product, methanol, which is further metabolized by the cell to produce multicarbon intermediates. Regulation of the sMMO system relies on the remarkable properties of an effector protein, protein B. This protein is capable of facilitating component interactions in the presence of substrate, modifying the redox potential of the diiron species at the active site. These interactions permit access of substrates to the hydroxylase, coupling electron transfer by the reductase with substrate oxidation and affecting the rate and regioselectivity of the overall reaction. The membrane-associated form is less well researched than the soluble enzyme, but is known to contain copper at the active site and probably iron. From an applied perspective, methanotrophs have enjoyed variable successes. Whole cells have been used as a source of single-cell protein (SCP) since the 1970s, and although most plants have been mothballed, there is still one currently in production. Our earlier observations that sMMO was capable of inserting an oxygen atom from dioxygen into a wide variety of hydrocarbon (and some non-hydrocarbon) substrates has been exploited to either produce value added products (e.g. epoxypropane from propene), or in the bioremediation of pollutants such as chlorinated hydrocarbons. Because we have shown that it is now possible to drive the reaction using electricity instead of expensive chemicals, there is promise that the system could be exploited as a sensor for any of the substrates of the enzyme.

Figures

Figure 1
Figure 1
Transmission electron micrographs of sections of type I and X and type II methane-oxidizing bacteria.
Figure 2
Figure 2
The catalytic synthesis of methanol from methane using the ICI (Imperial Chemical Industries) copper-based catalyst.
Figure 3
Figure 3
Energetics of methanol production from methane.
Figure 4
Figure 4
Model for the regulation of MMO in Methylosinus trichosporium OB3b in cells grown under high and low copper regimes. At high copper : biomass ratios, pMMO is derepressed and the hypothetical activator (A) and repressor (R) are bound to free copper (or some protein that strongly binds copper). Under low copper : biomass ratios, there is little copper (or its protein complex) to bind to the repressor, so R binds to repress pMMO transcription. The free activator can now bind to the upstream activating sequence (UAS) of mmoX to permit transcription of the sMMO-encoding genes (Murrell et al. 2000).
Figure 5
Figure 5
A computational model of the possible binding site for methane in the hydroxylase of sMMO. The hydrophobic residues forming the ‘horseshoe-shaped’ pocket are shown in green. The binuclear iron centre is in silver/grey, and the bound methane molecule is shown in blue (George et al. 1996).
Figure 6
Figure 6
Principal intermediates during the sMMO catalytic cycle. (References for each intermediate are given in the text.)
Figure 7
Figure 7
Structure of the hydroxylase from Methylococcus capsulatus (Bath) at 2.2 Å resolution (Rosenzweig et al. 1993). The subunits are coloured as follows: α are pale blue and green; β are royal blue and mid-blue and γ are yellow–green and yellow. The binuclear iron centres are represented by two orange spheres on the α subunits.
Figure 8
Figure 8
Surface diagram model showing the docking of protein B into the canyon on the hydroxylase (Walters et al. 1999). The α subunits of the hydroxylase are shown in red, β in blue and γ in yellow. Protein B has been translated away from its proposed docking site on the surface of the hydroxylase. Furthermore, protein B has been rotated 90° clockwise about the y-axis to expose the residues most involved in binding. The residues coloured blue are the most affected by binding.
Figure 9
Figure 9
Interaction between sMMO components.

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