NifEN: a versatile player in nitrogenase assembly, catalysis and evolution

The Mo-nitrogenase catalyzes the reduction of N₂ to NH₃ at the cofactor of its catalytic NifDK component. NifEN shares considerable homology with NifDK in primary sequence, tertiary structure and associated metallocenters. Better known for its biosynthetic function to convert an all-iron precursor (L-cluster; [Fe₈S₉C]) to a mature cofactor (M-cluster; [(R-homocitrate) MoFe₇S₉C]), NifEN also mimics NifDK in catalyzing substrate reduction at ambient conditions. The recently discovered ability of NifEN to reduce N₂ to NH₃ is particularly interesting, as it points to NifEN as a plausible, prototype ancient nitrogenase during evolution. Moreover, the dual function of NifEN in assembly and catalysis makes it a great template to reconstruct the functional variants or equivalents of NifDK, which could facilitate the mechanistic investigation and heterologous synthesis of nitrogenase. This perspective provides an overview of our recent studies of NifEN, with a focus on the implications of its functional versatility for nitrogenase assembly, catalysis and evolution.