How to handle copper

Bacteria that can convert methane could be incredibly useful, but in order for biotechnologists to take advantage of these methanotrophs we need to understand their ability to handle certain metals. 

Copper (Cu) is essential for most organisms, but can also be extremely toxic, and is safely handled using sensing, chaperoning, exporting and storage proteins. Bacteria are thought to have overcome the problems associated with Cu by limiting the use of this metal ion in the cytoplasm without the need for storage. However, an important group of bacteria called methanotrophs need large quantities of Cu to oxidise methane to methanol. Methane is produced by both geological and anthropogenic processes on Earth and is a greenhouse gas with a much greater warming potential than carbon dioxide. The consumption of methane by methanotrophs for carbon and energy is nature’s primary mechanism of limiting its release to the atmosphere. The availability of methane is increasing due to hydraulic fracturing of shale rock and methane can be obtained from sustainable sources, such as landfill waste sites. Using methane for the production of liquid fuels as well as bulk and fine chemicals is a current area of great interest². To unlock the potential of methanotrophs for the bio-conversion of methane requires a much better understanding of Cu-handling by these organisms.

The consumption of methane by methanotrophs for carbon and energy is nature’s primary mechanism of limiting its release to the atmosphere.

Almost all methanotrophs use the membrane-bound particulate methane monooxygenase (pMMO), which has Cu at its active site, to convert methane to methanol (some organisms have the ability to switch to the iron-requiring soluble sMMO when Cu levels are low). To satisfy their high demands for Cu, methanotrophs secrete a small modified Cu-sequestering peptide called methanobactin (mbtin) when Cu levels are low.

[caption id="attachment_53081" align="alignnone" width="400"] Structure of the Cu (I)-Csp1 tetramer, with the Cu(I) ions bound within each monomeric four-helix bundle shown as orange spheres[/caption]

The structures of mbtins and their ability to bind Cu with high affinity have been studied previously in our group³. To investigate the fate of Cu-mbtin in the model switchover methanotroph Methylosinus trichosporium OB3b, soluble extracts were analysed using metalloproteomic techniques. Mbtin could not be found, but a number of soluble Cu-containing fractions were observed. The most abundant of these contained a small, 122 amino acid, previously unstudied protein possessing 13 cysteines, residues that avidly bind Cu. Detailed analysis of this protein¹ demonstrated that it is a tetramer capable of binding up to 52 Cu(I) ions with high affinity, consistent with a role in the storage of Cu and was therefore named copper storage protein 1 (Csp1).

Each monomer of Csp1 is a four-helix bundle, whose core can be filled with 13 Cu(I) ions, bound mainly by the cysteines. Storing metal within a common protein fold is unprecedented. Mbtin can readily acquire Cu(I) from Csp1, and a mutant strain of M. trichosporium OB3b lacking Csp1 and the close homologue Csp2, switches to using sMMO more quickly than the wild type when Cu becomes limiting, consistent with a role for these proteins in Cu delivery to pMMO.

Each monomer of Csp1 is a four-helix bundle, whose core can be filled with 13 Cu(I) ions, bound mainly by the cysteines.

The discovery of the Csp proteins and their role in Cu storage is particularly timely given the recent resurgence of interest in using methanotrophs and MMOs in methane-related technologies. Bio-conversion could provide more cost effective methods to unlock the potential of methane as a feedstock, but requires a much better understanding of how methanotrophs metabolise, handle and utilise Cu for the conversion of methane to methanol.

The work published in Nature describing the discovery of the Csps1 adds important new insight into this complex process. Furthermore, intracellular Csps are widespread in a range of bacteria, an observation that challenges current ideas about how bacteria have adapted to cope with the potential toxicity of Cu.

Authors: Dr Nicolas Vita is a research associate at the Institute for Cell and Molecular Biosciences at Newcastle University.

Christopher Dennison is professor of Biological Chemistry at Newcastle University.

References:

1. N. Vita, S. Platsaki, A. Baslé, S. J. Allen, N. G. Paterson, A. T. Crombie, J. C. Murrell, K. J. Waldron and C. Dennison, A four-helix bundle stores copper for methane oxidation, Nature 2015, 525, 140-143. http://www.nature.com/nature/journal/v525/n7567/full/nature14854.html 2. C. A. Haynes and R. Gonzalez, Rethinking biological activation of methane and conversion to liquid fuels, Nature Chem. Biol. 2014, 10, 331-339. 3. A. El Ghazouani, A. Baslé, J. Gray, D. W. Graham, S. J. Firbank and C. Dennison, Variations in methanobactin structure influences copper utilization by methane-oxidizing bacteria, Proc. Natl. Acad. Sci. USA 2012, 109, 8400-8404.