Alternative nitrogenase activity in the environment and nitrogen cycle implications

Biological nitrogen fixation, the main natural input of fixed nitrogen into the biosphere, is catalyzed by Mo-, V-, or Fe-only nitrogenase metalloenzymes. Although “alternative” V- and Fe-only nitrogenase genes are found in many environments, the contribution of these isoenzymes to N₂ fixation is unknown. Here we present a new method (ISARA, isotopic acetylene reduction assay) that distinguishes canonical Mo and alternative nitrogenase activities based on in vivo ¹³C fractionation of acetylene reduction to ethylene (¹³ε_Mo = 13.1–14.7 ‰, ¹³ε_V = 7.5–8.8 ‰, ¹³ε_Fe = 5.8–6.5 ‰). ISARA analyses indicate significant contributions of alternative nitrogen fixation in boreal cyanolichens and salt marshes (~10–40 % acetylene reduction, ~20–55 % N₂ fixed). These results affect the quantitative interpretation of natural abundance ¹⁵N data or traditional acetylene reduction assays. They also invite a reexamination of the conditions under which the different nitrogenase isozymes are active and suggest significant interactions between the cycles of nitrogen and trace metals.