The essential role of copper in bacterial methane oxidation: characterising a novel copper protein involved in storage and the soluble copper proteome

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Methane-oxidising bacteria use methane, a potent greenhouse gas, as a source of carbon and energy. To enable these organisms to perform this key function most effectively they require large quantities of copper. Methane-oxidising bacteria have evolved novel mechanisms that ensure they are able to accumulate the copper they need to metabolise methane. This includes the secretion of small peptides called methanobactins (mbtins) that are able to sequester the minute amounts of copper present in the organism's surrounding environment. Methane-oxidising bacteria use mbtin to uptake copper, with one of the main uses being for the enzyme that oxidises methane (a methane monooxygenase). How these organisms handle copper, which is potentially toxic as well as being essential, has not been studied in any detail. Whilst analysing extracts from Methylosinus trichosporium OB3b, a methane-oxidising bacterium, for mbtin we have found a number of high abundance soluble copper pools containing proteins that bind this metal. The presence of large amounts of soluble copper in M. trichosporium OB3b was unanticipated as only two low-abundance soluble copper proteins are predicted (the copper-containing methane monooxygenase is membrane bound). One of the high abundance copper pools has been found to contain a novel copper protein, potentially involved in storing copper ions, herein called Csp1. The other soluble copper pools have not yet been studied in any detail.In the proposed work we will study copper binding to Csp1 both in M. trichosporium OB3b and also using protein that we have produced in large amounts via an over-expression host. We will analyse the structure of Csp1 and how this is influenced by copper binding. We will investigate the role of Csp1 in copper storage by genetically engineering M. trichosporium OB3b, as well as by looking at how copper levels during growth influence the production of Csp1. Proteins similar to Csp1 are present in methane-oxidising bacteria and also in other bacteria. The Csp1 homologues from M. trichosporium OB3b and Bacillus subtilis, an organism in which copper handling has been studied and that is more tractable for genetic engineering approaches, will be studied. The other soluble copper pools that have been identified in M. trichosporium OB3b will also be analysed with the aim of finding additional novel copper-binding proteins. The proposed studies will provide insight into copper management in a relatively simple system of environmental importance and therefore has applications in helping to understand the oxidation of methane in Nature and the mitigation of the contribution this greenhouse gas makes to global warming. Furthermore, vast gaseous methane reserves are untapped as a feedstock for liquid fuels and chemical production due to the expense and difficulties associated with the available processes that can facilitate the conversion to methanol, a reaction readily performed by methane-oxidising bacteria. Understanding how these organisms handle an essential cofactor, copper, for the oxidation of methane therefore has potential biotechnological applications.