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ABSTRACT 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 (CO 2 ) into the potent greenhouse gas methane (CH 4 ). Here, we report carbon ( 13 C/ 12 C) and hydrogen ( 2 H/ 1 H) 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 ( 13 α CO2/CH4 = 1.051 ± 0.002 for V-nitrogenase and 1.055 ± 0.001 for Fe-only nitrogenase; values are means ± standard errors). In contrast, the hydrogen isotope fractionations ( 2 α H2O/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 2 α H2O/CH4 shows that the reaction pathway nitrogenases use to form methane strongly discriminates against 2 H, and that 2 α H2O/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. 

Biological nitrogen fixation is catalyzed by the molybdenum (Mo), vanadium (V) and iron (Fe)‐only nitrogenase metalloenzymes. Studies with purified enzymes have found that the ‘alternative’ V‐ and Fe‐nitrogenases generally reduce N₂more slowly and produce more byproduct H₂than the Mo‐nitrogenase, leading to an assumption that their usage results in slower growth. Here we show that, in the metabolically versatile photoheterotrophRhodopseudomonas palustris, the type of carbon substrate influences the relative rates of diazotrophic growth based on different nitrogenase isoforms. The V‐nitrogenase supports growth as fast as the Mo‐nitrogenase on acetate but not on the more oxidized substrate succinate. Our data suggest that this is due to insufficient electron flux to the V‐nitrogenase isoform on succinate compared with acetate. Despite slightly faster growth based on the V‐nitrogenase on acetate, the wild‐type strain uses exclusively the Mo‐nitrogenase on both carbon substrates. Notably, the differences in H₂:N₂stoichiometry by alternative nitrogenases (~1.5 for V‐nitrogenase, ~4–7 for Fe‐nitrogenase) and Mo‐nitrogenase (~1) measured here are lower than priorin vitroestimates. These results indicate that the metabolic costs of V‐based nitrogen fixation could be less significant for growth than previously assumed, helping explain why alternative nitrogenase genes persist in diverse diazotroph lineages and are broadly distributed in the environment.