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

Significance Methane emission from the ruminant livestock sector—a by-product from enteric fermentation of plant biomass in the ruminant digestive system—is produced by methanogenic archaea and represents not only a significant amount of anthropogenic greenhouse gases contributing to climate change but also an energy loss and a reduction in feed efficacy. The present study elucidates the development and the unique mode of action of the highly specific inhibitor 3-nitrooxypropanol (3-NOP), which is targeting the nickel enzyme methyl-coenzyme M reductase in rumen archaea that catalyzes the methane-forming reaction. At the very low effective concentrations recently applied in vivo (dairy and beef cattle), 3-NOP appears to inhibit only methanogens and thus to be attractive for development as a feed supplement. 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