Microbes known as methanogenic archaebacteria produce around 90% of the atmosphere’s methane.
These tiny organisms, which reside in extreme environments, like hot geysers and sulfurous lakes, as well as deep under the ocean, are able to produce methane as a metabolic byproduct under anerobic conditions.
The mechanisms by which they do this has been well studied, but a central question has remained unanswered for decades.
The enzyme that catalyzes the final step of methane production in archaebacteria is called methyl coenzyme M reductase (MCR). The reaction takes two substrates: a methyl donor called methyl coenzyme M (CH3-SCoM) and an electron donor called coenzyme B (CoB-SH).
During the reaction, methane (CH4) is produced when the methyl group (CH3) from CH3-ScoM acquires a hydrogen atom from CoB-SH. What scientists have been unable to agree upon is the precise mechanism by which this happens and which intermediates are formed along the way.
Two possibilities have been put forward, which both center on the interaction of the substrates with a critical Nickel ion at the enzyme’s active site. The first (mechanism 1) says that a methyl-nickel intermediate forms, when the methyl group from CH3-SCoM bonds to the Nickel ion.
The second (mechanism 2) says that a methyl radical breaks away from CH3-SCoM after the sulfur atom in the coenzyme bonds to the Nickel ion.
But scientists have been unable to agree which of the two proposed mechanisms is correct because the intermediate products of the reaction have never been observed because of their extremely transient nature.
A recent study, published in Science, may have finally laid the debate to rest. The researchers replicated the reaction that takes place in archaebacteria by mixing MCR with CH3-SCoM and adding a modified version of CoB-SH. This modified coenzyme slowed the reaction down, allowing the intermediates to accumulate.
The researchers, led by Stephen Ragsdale from the University of Michigan, studied the reaction using electron paramagnetic resonance (EPR) spectroscopy. EPR was the ideal method to carry out the research, as the only technique to unambiguously identify unpaired electrons.
The intermediate produced via mechanism 1 contains an unpaired electron which should be detectable via EPR, while the intermediate formed via mechanism 2 should be EPR-silent, giving the researchers a straightforward way to distinguish between the two.
In their experiments, Ragsdale et al. used a Bruker EPR device. EPR is an invaluable method for the detection of metalloproteins, such as the nickel-containing MCR enzyme.
The team used a method known as rapid freeze-quench EPR which allowed them study the reaction at multiple time points like a freeze-frame. Their results showed that the signal from active MCR, which is also EPR-visible, deintensified as the reaction progressed.
But if mechanism 1 were true, they would expect to see a comparable increase in a signal from an EPR-visible intermediate. No such signal was observed. Instead the results support mechanism 2 – the presence of an EPR-silent intermediate.
Taken together with other evidence from magnetic circular dichroism, computational, and experimental thermodynamic analyses, the researchers say the findings strongly favor mechanism 2.
They explain the finding is surprising because the methyl radical is so highly reactive. The results mean that the MCR enzyme must be extremely specific in the way it positions the substrates to ensure the methyl radical only reacts with one specific hydrogen atom located on coenzyme B.
Future research will explore the reverse reaction catalyzed by MCR – methane oxidation – as well as how biomimetic strategies could be used to harness the MCR mechanism in the generation and activation of methane.
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- 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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