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X-ray absorption spectroscopy, XAS, has been used to probe the metal centre of an important enzyme that can oxidise methane, natural gas, to methanol. Turns out the metal is copper not iron as previously thought and the discovery could open up a route to novel catalysts for converting "waste" methane (either from landfill or oil well flare-off) into useful liquid methanol for processing into fuel or other more valuable products. Current technological limitations mean that it is economically unviable to exploit the vast natural gas resources available to us by converting the methane to methanol and thence into liquid fuels and chemical feedstock materials. There are just no sustainable ways to oxidise methane as almost always developments fail in terms of the high temperatures and pressures required for the reaction or else their inefficiencies lead to untenable amounts of waste by-products. Nature, of course, has evolved an optimal solution to methane oxidation in the form of metalloenzymes known as methane mono-oxygenases (MMOs) used by methanotrophic bacteria. MMOs can oxidise methane under ambient conditions in what from a chemical engineering perspective is a highly efficient and environmentally friendly way. Such a catalyst would be a boon while humanity continues to rely on carbon sources for fuel and heating. Ramakrishnan Balasubramanian, Stephen Smith, Liliya Yatsunyk of the Department of Biochemistry, Molecular Biology and Cell Biology and the Department of Chemistry, at Northwestern University, in Evanston, Illinois, working with Swati Rawat and Timothy Stemmler of the Department of Biochemistry and Molecular Biology, at Wayne State University, in Detroit, Michigan, USA, have focused their research on MMOs with a view to developing a viable catalyst for industrial methane oxidation. There are two types of MMO known. The first: soluble MMOs are produced by various strains of methanotrophic bacteria when copper is scarce. These have a well-characterised catalytic site with iron at its core. The second form is particulate MMO (pMMO), which is a membrane-bound metalloenzyme produced by all methanotrophs. This latter form has been known for twenty years and is comprised of three subunits, pmoA, pmoB and pmoC, hooked together in a trimeric unit. However, two crystal structures have failed to reveal definitively the metal composition and location of the active site in pMMO. Balasubramanian have now demonstrated that the activity of pMMO, from Methylococcus capsulatus, is dependent on copper, rather than iron as suggested by Mossbauer spectroscopy, and that the copper active site is located in the soluble domains of the pmoB subunit rather than within the membrane as revealed by probing with X-ray absorption spectroscopy. The researchers explain that recombinant forms of the soluble fragments of pmoB can bind to copper and retain methane oxidation activity, as well as being able to oxidise a higher hydrocarbon, propylene. When they used mutagenesis to disrupt the copper centres in soluble pmoB their results indicated that the active site contains a dicopper centre. "These findings help resolve the pMMO controversy and provide a promising new approach to developing environmentally friendly C-H oxidation catalysts," the team says. "Our work shows that pMMO is a copper enzyme and that the active site is located in the N-terminal soluble domain of the pmoB subunit at the site of the crystallographic dicopper centre," the team concludes. "Neither the proposed intramembrane di-iron centre nor the proposed intra- membrane tricopper centre is the active site. The copper stoichiometry, XAS and activity data are most consistent with a dicopper, rather than a tricopper, active site." They point out that given that the soluble pmoB proteins have lower activity than particulate MMO and isolated pMMO less activity than a whole cell system, the next step will be to determine which additional subunits and trimeric structure are required for a fully active enzyme, which will hopefully lead the research to the next phase of developing a hydrocarbon oxidising catalyst.
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Metals at the heart of MMO |
