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EPR Elucidates Microbial Methane Production

Methane-producing microbes, known as methanogenic archaebacteria, account for around 90% of all methane in the Earth’s atmosphere. But despite being well studied, a mystery has remained over the precise mechanism by which they produce the compound, a greenhouse gas many times more potent than carbon dioxide. Using a Bruker electron paramagnetic resonance (EPR) instrument researchers at the University of Michigan have recently helped settle this 25-year long debate.

An unsolved equation

The key enzyme involved in the production of methane by archaebacteria is called methyl coenzyme M reductase (MCR). A defining feature of the enzyme is a nickel ion at its active site that is involved in the reaction but, until now, scientists have been unable to completely agree how.

The enzyme catalyzes the final step of methane production by taking two substrates, methyl-coenzyme M (CH3-SCoM) and coenzyme B (CoB-SH), and converting them to methane and another compound called heterodisulfide (CoM-S-S-CoB). During the reaction, methane (CH4) is produced when a methyl group (CH3) from CH3-SCoM acquires an additional hydrogen from CoB-SH. There are two possible intermediate mechanisms that lead to this happening:

1) Via the formation of a methyl-nickel intermediate. In this case, the methyl group from CH3-ScoM becomes transiently bound to the nickel ion in the enzyme’s active site.

2) Via the formation of a methyl radical intermediate. In this case ScoM becomes transiently bound to the nickel ion in the enzyme’s active site, while the methyl group breaks away as a radical.

The two intermediates should be distinguishable via EPR spectroscopy because the methyl-nickel intermediate formed by mechanism 1 contains an unpaired electron, while the intermediate formed by mechanism 2 is EPR-silent. However, the intermediates produced during the reaction have never been identified because it proceeds so fast and they exist only transiently. As a result, scientists have been unable to definitively agree which mechanism is correct.

Slow-motion vision

In a study published in Science, a team of researchers found a way to overcome this obstacle and perhaps finally lay the debate to rest.

The team, led by Stephen Ragsdale from the University of Michigan, studied the reaction using a shortened version of CoB-SH. This modification slows the reaction rate down, allowing the intermediate products to accumulate and be observed.

First, the researchers studied the speed of the reaction using EPR. They did this by comparing the rate of disappearance of the active MCR enzyme, which is detectable by EPR, with the rate of methane production. They found that these rates were virtually identical. This was their first support for mechanism 2 over mechanism 1.

Mechanism 2 involves the production of an unstable methyl radical which would react almost immediately to obtain the hydrogen atom required to form a stable molecule. By contrast, mechanism 1 involves the generation of a more stable methyl-nickel complex, which requires an additional step for the production of methane, meaning this should occur at a slower rate than the disappearance of active MCR.

But the researchers wanted to obtain more direct evidence of the intermediates. To do this, they used a method called rapid freeze-quench EPR. This allowed them to take snapshots during the first minute of the reaction at multiple time points. These results showed a decrease in intensity of the signal from active MCR at a rate comparable to methane synthesis. But there was no simultaneous appearance of an EPR-detectable intermediate that could be attributed to the formation of a methyl-nickel complex, as would be expected with mechanism 1. Instead, the presence of an EPR-silent intermediate provided clear evidence supporting mechanism 2.

Furthermore, the team showed that spectra derived from the same samples using magnetic circular dichroism, as well as computation, and experimental thermodynamic analyses, also strongly favored mechanism 2. The evidence from this research, which unequivocally dismisses mechanism 1, should be the first step to establishing a consensus on the mechanism of MCR in methane production.

But it is not just about settling scores. Thanks to EPR, the findings have major implications for our ability to synthetically generate and activate methane. The researchers suggest that using a biomimetic strategy could allow the creation of catalysts that can convert methane into liquid fuels. This could be important for energy sustainability given that reserves of natural gas are increasing more rapidly than reserves of petroleum, as well as offering the potential to harness anthropogenic sources of methane, which contribute to around 20% of the world’s annual greenhouse gas warming potential.

References

  • Chen S-L, Blomberg MRA & Siegbahn PEM. How Is Methane Formed and Oxidized Reversibly When Catalyzed by Ni-Containing Methyl-Coenzyme M Reductase? Chemistry – A European Journal 2012; 18: 6309-6315.
  • Lawton TJ, Rosenzweig AC. Methane – make it or break it. Science 2016; 352: 892-893.
  • Lunsford JH. Catalytic conversion of methane to more useful chemicals and fuels: a challenge for the 21st century. Catalysis Today 2000; 63: 165-174.
  • Scheller S, Goenrich M, Mayr S, et al. Intermediates in the Catalytic Cycle of Methyl Coenzyme M Reductase: Isotope Exchange is Consistent With Formation of a s-Alkane–Nickel Complex. Angewandte Chemie International Edition 2010; 49: 8112-8115.
  • Wongnate T, Sliwa D, Ginovska B, et al. The radical mechanism of biological methane synthesis by methylcoenzyme M reductase. Science 2016; 352: 953-958.

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