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

Abstract

Ruminants, such as cows, sheep, and goats, predominantly ferment in their rumen plant material to acetate, propionate, butyrate, CO2, 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.

Keywords: climate change; enteric methane; greenhouse gas; methanogenesis; methyl-coenzyme M reductase.

Fig. 1.

Methane formation in the rumen of a dairy cow and its inhibition by 3-nitrooxypropanol (3-NOP). The H₂ concentration in the rumen fluid is near 1 µM (≙140 Pa = 0.14% in the gas phase).

Fig. 2.

Binding of 3-NOP to methyl-coenzyme M reductase (MCR) as suggested by molecular docking. The crystal structure of inactive isoenzyme I from M. marburgensis was used in the docking experiments (25). (A) MCR-catalyzed reaction. CH₃-S-CoM, methyl-coenzyme M; CoM-S-S-CoB, heterodisulfide of coenzyme M and coenzyme B; HS-CoB, coenzyme B. (B) 3-NOP in the active site with its nitrate group in electron transfer distance to Ni(I) of F₄₃₀ and its hydroxyl group interacting via a water molecule with Arg120. (C) 3-NOP in the active site with its hydroxyl group in coordination distance to Ni(I) of F₄₃₀ and its nitrate group interacting with Arg120. (D) Methyl-coenzyme M (CH₃-S-CoM) in the active site with its thioether sulfur in electron transfer distance to Ni(I) and its sulfonate group interacting with both a water molecule and Arg120. The molecules 3-NOP, CH₃-S-CoM, and coenzyme B (HS-CoB) are drawn as ball-and-stick models in orange and F₄₃₀ in light gray highlighting nitrogen in blue, oxygen in red, sulfur in yellow, and nickel(I) as a green sphere. The position of methyl-coenzyme M obtained via docking is almost identical to that found via EPR measurements of active MCR (28).

Fig. 3.

Inactivation of MCR by 3-NOP and nitrite. The experiments were performed with purified isoenzyme I from Methanothermobacter marburgensis. (A) Effect of 3-NOP on the MCR activity; (B) Effect of 3-NOP on the EPR signals MCR_red1 (78 µM) and MCR_ox1 (4 µM) (30). The spectrum (Inset) remaining after complete MCR inactivation with 3-NOP (100 µM) is that of MCR_ox1 (see In Vitro Studies). (C) Formation of nitrite and nitrate upon inactivation of MCR with 3-NOP (50 mM). (D) Effect of nitrite and nitrate on the EPR signal MCR_red1. The 0.3-mL assays contained about 1 mM coenzyme M that was added to MCR during purification and storage to stabilize its activity (Methods).

Fig. 4.

View into the active site of MCR crystallized before (A) and after (B) in vivo inactivation by 3-NOP. The crystal structures were resolved to 1.25-Å resolution. Coenzyme B (HS-CoB) (at an occupancy near 100%) and coenzyme M (HS-CoM) (at an occupancy near 80%) are drawn as ball-and-stick models in orange and F₄₃₀ in light gray with nickel highlighted as a green sphere. The secondary structure of the protein is cartooned in light purple. Three water molecules are shown as red spheres. The 2F_o-mF_c map is contoured at 1σ in dark-blue mesh. For an interpretation, see In Vitro Studies.

Fig. 5.

Inhibition of growth of two methanogenic archaea on H₂ (80%) and CO₂ (20%) in the presence of 3-NOP. (A) Methanothermobacter marburgensis grown at 65 °C in a 25-mL batch culture with a 100-mL gas phase; (B) Methanobrevibacter ruminantium grown at 37 °C in a 5-mL batch culture with a 10-mL gas phase.

Fig. S1.

Conversion of 3-NOP to 1,3-propanediol in ex situ rumen fluid under strictly anoxic conditions. The gas phase was 100% CO₂, and the temperature was 38 °C. The rumen fluid (0.4 mL) was diluted with 1.6 mL of buffer A (pH 6.5) and supplemented with 0.05 g of feed dry matter (50% grass silage and 50% compound feed). At time 0, 3-nitrooxy[3-¹⁴C]propane-1-ol was added (0.036 µmol/10 mL, 28 µCi/µmol), and after the time intervals indicated, samples were analyzed for ¹⁴C radioactivity via radio-HPLC. The radioactivity cochromatographed with 3-NOP (21-min retention time) and with 1,3-propanediol (5-min retention time). No conversion was observed under aerobic conditions. For quantitative analysis, peak integration was performed with the Berthold Radiostar 4.6.0.0 software. Peaks in the radioactivity chromatogram were integrated after background subtraction. The sum of integrated peaks were set to 100%. ROI, region of interest.