Large hydrogen isotope fractionation distinguishes nitrogenase-derived methane from other methane sources
Biological nitrogen fixation is catalyzed by the enzyme nitrogenase. Two forms of this metalloenzyme, the vanadium (V)-and iron (Fe)-only nitrogenases, were recently found to reduce small amounts of carbon dioxide (CO2) into the potent greenhouse gas methane (CH4). Here, we report carbon (13C/12C) and hydrogen (2H/1H) stable isotopic compositions and fractionations of methane generated by Vand Fe-only nitrogenases in the metabolically versatile nitrogen fixer Rhodopseudomonas palustris. The stable carbon isotope fractionation imparted by both forms of alternative nitrogenase are within the range observed for hydrogenotrophic methanogenesis (13αCO2/CH4 = 1.051±0.002 for V-nitrogenase and 1.055±0.001 for Feonly nitrogenase; values are means ± standard errors). In contrast, the hydrogen isotope fractionations (2αH2O/CH4 = 2.071±0.014 for V-nitrogenase and 2.078±0.018 for Fe-only nitrogenase) are the largest of any known biogenic or geogenic pathway. The large 2αH2O/CH4 shows that the reaction pathway nitrogenases use to form methane strongly discriminates against 2H, and that 2αH2O/CH4 distinguishes nitrogenase-derived methane from all other known biotic and abiotic sources. These findings on nitrogenase-derived methane will help constrain carbon and nitrogen flows in microbial communities and the role of the alternative nitrogenases in global biogeochemical cycles. IMPORTANCE All forms of life require nitrogen for growth. Many different kinds of microbes living in diverse environments make inert nitrogen gas from the atmosphere bioavailable using a special enzyme, nitrogenase. Nitrogenase has a wide substrate range, and, in addition to producing bioavailable nitrogen, some forms of nitrogenase also produce small amounts of the greenhouse gas methane. This is different from other microbes that produce methane to generate energy. Until now, there was no good way to determine when microbes with nitrogenases are making methane in nature. Here, we present an isotopic fingerprint that allows scientists to distinguish methane from microbes making it for energy versus those making it as a by-product of nitrogen acquisition. With this new fingerprint, it will be possible to improve our understanding of the relationship between methane production and nitrogen acquisition in nature. © 2020 Luxem et al.
Applied and Environmental Microbiology