Large hydrogen isotope fractionations distinguish nitrogenase-derived methane from other sources

Nitrogenase is the main source of natural fixed nitrogen for the biosphere. Two forms of this metalloenzyme, the vanadium (V) and iron (Fe)-only nitrogenases, were recently found to reduce small amounts of carbon dioxide into the potent greenhouse gas methane. Here we report carbon and hydrogen stable isotopic compositions and fractionations of methane generated by V- and 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 (13CO2/CH4 = 1.051 {+/-} 0.002 for V-nitrogenase and 1.055 {+/-} 0.001 for Fe-only nitrogenase, mean {+/-} SE). In contrast, the hydrogen isotope fractionations (2H2O/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 2H2O/CH4 shows that the reaction pathway nitrogenases use to form methane strongly discriminates against 2H, and that 2H2O/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. ImportanceAll 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 protein, 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 developed an isotopic fingerprint that allows scientists to distinguish methane from microbes making it for energy versus those making it as a byproduct 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.