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Methods pinpoint copper binding sites in enzyme from methane-munching bacteria

Two studies suggest methane monooxygenase has two sites that each bind one copper ion

by Celia Henry Arnaud
June 26, 2019 | A version of this story appeared in Volume 97, Issue 26


A ribbon structure of a bacterial methane monooxygenase showing the location of the copper binding sites
Credit: Nat. Commun.
This methane monooxygenase's copper binding sites are located in the PmoB (purple) and PmoC (blue) subunits. PmoA is shown in pink.

A pair of recent papers helps clear up a longstanding mystery about the enzyme particulate methane monooxygenase (pMMO). The enzyme sits in the membranes of bacteria that consume methane as their main food source, and catalyzes the conversion of methane to methanol.

“Everyone in the field agrees that it’s a copper-dependent enzyme,” says Amy C. Rosenzweig, a biochemist at Northwestern University who led both studies. What researchers don’t agree on is what the copper active site might look like. Decades of conflicting studies have left the active site shrouded in mystery. Various crystal structures have shown zinc, copper, or no metal at all in the potential binding sites.

In a study published in May, Rosenzweig, Brian M. Hoffman, and their coworkers used multiple electron paramagnetic resonance (EPR) spectroscopic techniques to reveal that pMMO purified from bacterial membranes contains two metal binding sites, each of which binds a single copper ion (Science 2019, DOI: 10.1126/science.aav2572). Rosenzweig points out that the team had previously proposed that one of the sites had a dicopper center.

For one of the two sites, the researchers couldn’t isolate its EPR signal, preventing them from characterizing the site. All they could tell was how far its copper ion was from the other site’s copper ion—about 2 nm. Previous crystal structures were no help because those data had shown that the mystery site was in a chronically disordered region of the protein.

So Rosenzweig turned to Neil L. Kelleher, another Northwestern chemistry professor, for help. Kelleher used top-down native mass spectrometry to analyze the protein. In that technique, researchers reconstitute the protein in a lipid nanodisc under conditions that cause it to remain in its native state.

Members of Kelleher’s team were able to determine the number of coppers in each of pMMO’s three subunits, named PmoA, PmoB, and PmoC (Nat. Commun. 2019, DOI: 10.1038/s41467-019-10590-6). They found a single copper binding site in each of PmoB (the one that was visible with EPR) and PmoC (the one that was obscured).

The mass spec experiment told the team something else about the enzyme. While the researchers prepared the enzyme samples for native top-down mass spectrometry, the protein lost some of its copper. By adding copper during the preparation, the researchers could help the enzyme recover the metal, particularly in PmoC. The added copper also increased the enzyme’s activity, suggesting that the PmoC site is important for enzyme activity. But questions remain about what the active site looks like.

“The fact that copper is located in subunit PmoC and correlates with activity is something of a surprise and could indicate the location of the active site,” says Thomas J. Smith, an expert on pMMO at Sheffield Hallam University. “There is still such a large amount of conflicting evidence about the location of the active center that I think additional evidence is still needed.”


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