Abstract
Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the rate-limiting and final step in methane biosynthesis. Using coenzyme B as the two-electron donor, MCR reduces methyl-coenzyme M (CH3-SCoM) to methane and the mixed disulfide, CoBS-SCoM. MCR contains an essential redox-active nickel tetrahydrocorphinoid cofactor, Coenzyme F430, at its active site. The active form of the enzyme (MCRred1) contains Ni(I)-F430. Rapid and efficient conversion of MCR to MCRred1 is important for elucidating the enzymatic mechanism, yet this reduction is difficult because the Ni(I) state is subject to oxidative inactivation. Furthermore, no in vitro methods have yet been described to convert Ni(II) forms into MCRred1. Since 1991, it has been known that MCRred1 from Methanothermobacter marburgensis can be generated in vivo when cells are purged with 100% H2. Here we show that purging cells or cell extracts with CO can also activate MCR. The rate of in vivo activation by CO is about 15 times faster than by H2 (130 and 8 min-1, respectively) and CO leads to twofold higher MCRred1 than H2. Unlike H2-dependent activation, which exhibits a 10-h lag time, there is no lag for CO-dependent activation. Based on cyanide inhibition experiments, carbon monoxide dehydrogenase is required for the CO-dependent activation. Formate, which also is a strong reductant, cannot activate MCR in M. marburgensis in vivo.
Highlights
Methanogens are responsible for all biological methane production on earth, generating 109 tons of methane annually
GENERATION OF MCRred1 IN VIVO BY CO To date, the most effective way to activate MCR has been to incubate the cell suspension under a H2 atmosphere (Rospert et al, 1991a; Kunz et al, 2006). While this is effective for generating MCRred1 from organisms like M. marburgensis, for some methanogens, like Methanosarcina acetivorans, H2 activation is inefficient because the hydrogenases are weakly expressed (Guss et al, 2009; Wang et al, 2011)
The activity of MCR is linearly related to the MCRred1 electron paramagnetic resonance (EPR) signal, with g values at 2.24 and 2.065; EPR spectroscopy is a convenient method to assess the level of MCR activation achieved
Summary
Methanogens are responsible for all biological methane production on earth, generating 109 tons of methane annually. Methanogens play critical roles in the carbon cycle by converting products from anaerobic fermentation (such as hydrogen, carbon dioxide, methanol, formate, and acetate) into methane (Thauer, 1998; Ferry, 2010). By such means can methanogens obtain energy to grow (Thauer, 1998; Ferry, 2010). Because of the effect of greenhouse gases on climate change, understanding the basis of methane production is an important research goal, while controlling methane emissions is an important aim for governmental policy makers
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