Ferredoxin-dependent methane formation from acetate in cell extracts of Methanosarcina barkeri (strain MS)

AbstractCell extracts of Methanosarcina barkeri grown on acetate catalyzed the conversion of acetyl-CoA to CO2 and CH4 at a specific rate of 50 nmol·min−1·mg−1. When ferredoxin was removed from the extracts by DEAE-Sephacel anion exchange chromatography, the extracts were inactive but full activity was restored upon addition of purified ferredoxin from M. barkeri or from Clostridiwn pasteurianum. The apparent Km for ferredoxin from M. barkeri was determined to be 2.5 μM. A ferredoxin dependence was also found for the formation of CO2, H2 and methylcoenzyme M from acetyl-CoA, when methane formation was inhibited by bromoethanesulfonate. Reduction of methyl-coenzyme M with H2 did not require ferredoxin. These and other data indicate that ferredoxin is involved as electron carrier in methanogenesis from acetate. Methanogenesis from acetyl-CoA in cell extracts was not dependent on the membrane fraction, which contains the cytochromes

Mode of action uncovered for the specific reduction of methane emissions from ruminants by the small molecule 3-nitrooxypropanol

Ruminants, such as cows, sheep, and goats, predominantly ferment in their rumen plant material to acetate, propionate, butyrate, CO, and methane. Whereas the short fatty acids are absorbed and metabolized by the animals, the greenhouse gas methane escapes via eructation and breathing of the animals into the atmosphere. Along with the methane, up to 12% of the gross energy content of the feedstock is lost. Therefore, our recent report has raised interest in 3-nitrooxypropanol (3-NOP), which when added to the feed of ruminants in milligram amounts persistently reduces enteric methane emissions from livestock without apparent negative side effects [Hristov AN, et al. (2015) Proc Natl Acad Sci USA 112(34):10663-10668]. We now show with the aid of in silico, in vitro, and in vivo experiments that 3-NOP specifically targets methyl-coenzyme M reductase (MCR). The nickel enzyme, which is only active when its Ni ion is in the+1 oxidation state, catalyzes the methane-forming step in the rumen fermentation. Molecular docking suggested that 3-NOP preferably binds into the active site of MCR in a pose that places its reducible nitrate group in electron transfer distance to Ni(I). With purified MCR, we found that 3-NOP indeed inactivates MCR at micromolar concentrations by oxidation of its active site Ni(I). Concomitantly, the nitrate ester is reduced to nitrite, which also inactivates MCR at micromolar concentrations by oxidation of Ni(I). Using pure cultures, 3-NOP is demonstrated to inhibit growth of methanogenic archaea at concentrations that do not affect the growth of nonmethanogenic bacteria in the rumen.We thank Peter Livant (Auburn University), Elisabeth Jimenez (Consejo Superior de Investigaciones Cientificas), John Wallace (University of Aberdeen), and Jamie Newbold (Aberystwyth University) for the use of the gas chromatograph, for the in vitro culture work, and for providing stock pure cultures, respectively; Ulrich Ermler and the staff of the PXII beam line at Swiss Light Source (Villigen, Switzerland) for helping with the X-ray data collection; and David Rinaldo (Schrödinger, LLC) for molecular modeling support.Peer Reviewe

Single step purification of methylenetetrahydromethanopterin reductase from Methanobacterium thermoautotrophicum by specific binding to Blue Sepharose CL-6B

AbstractMethylenetetrahydromethanopterin reductase from metanogenic archaebacteria catalyzes the reversible reduction of N5,N10-methylenetetrahydro-methanopterin to N5-methyltetrahydromethanopterin with reduced coenzyme F420 as electron donor. The enzyme is involved in methane formation from CO2, and in methanoi disproportionation to CO2 and CH4. We report here that the reductase from Methanobacterium thermoautotrophicum specifically binds to Blue Sepharose CL-6B. Binding was competitive with coenzyme F420 rather than with NAD, NADP, FAD, FMN, AMP, ADP and ATP. The reductase could also be desorbed with salt. Based on this property an affinity Chromatographie procedure for the purification of the enzyme was developed